Therapeutic uses of inhibitors of RTP801

ABSTRACT

The present invention provides novel molecules, compositions, methods and uses for treating microvascular disorders, eye diseases and respiratory conditions based upon inhibition of the RTP801 gene and/or protein.

This application claims priority of EP patent application No. EP04019405.2, filed 16 Aug. 2004; U.S. provisional applications Nos.60/601,983, filed 17 Aug. 2004; 60/604,668, filed 25 Aug. 2004;60/609,786, filed 14 Sep. 2004; 60/638,659, filed 22 Dec. 2004;60/664,236, filed 22 Mar. 2005 and 60/688,943, filed 8 Jun. 2005, all ofwhich are hereby incorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to novel siRNA molecules which inhibit theRTP801 gene and to the use of such molecules to treat respiratorydisorders of all types (including pulmonary disorders), eye diseases andconditions, microvascular disorders, angiogenesis- and apoptosis-relatedconditions.

BACKGROUND OF THE INVENTION

Chronic Obstructive Pulmonary Disease (COPD)

Chronic obstructive pulmonary disease (COPD), affects more than 16million Americans and is the fourth highest cause of death in the UnitedStates. Cigarette smoking causes most occurrences of the debilitatingdisease but other environmental factors cannot be excluded (Petty T L.2003. Definition, epidemiology, course, and prognosis of COPD. Clin.Cornerstone, 5-10).

Pulmonary emphysema is a major manifestation of COPD. Permanentdestruction of peripheral air spaces, distal to terminal bronchioles, isthe hallmark of emphysema (Tuder R M, et al Oxidative stress andapoptosis interact and cause emphysema due to vascular endothelialgrowth factor blocade. Am J Respir Cell Mol Biol, 29:88-97; 2003.).Emphysema is also characterized by accumulation of inflammatory cellssuch as macrophages and neutrophils in bronchioles and alveolarstructures (Petty, 2003).

The pathogenesis of emphysema is complex and multifactorial. In humans,a deficiency of inhibitors of proteases produced by inflammatory cells,such as alpha1-antitrypsin, has been shown to contribute toprotease/antiprotease imbalance, thereby favoring destruction ofalveolar extracellular matrix in cigarette-smoke (CS) induced emphysema(Eriksson, S. 1964. Pulmonary Emphysema and Alpha1-AntitrypsinDeficiency. Acta Med Scand 175:197-205. Joos, L., Pare, P. D., andSandford, A. J. 2002. Genetic risk factors of chronic obstructivepulmonary disease. Swiss Med Wkly 132:27-37). Matrix metalloproteinases(MMPs) play a central role in experimental emphysema, as documented byresistance of macrophage metalloelastase knockout mice against emphysemacaused by chronic inhalation of CS (Hautamaki, et al: Requirement formacrophage elastase for cigarette smoke-induced emphysema in mice.Science 277:2002-2004). Moreover, pulmonary overexpression ofinterleukin-13 in transgenic mice results in MMP- andcathepsin-dependent emphysema (Zheng, T., et al 2000. Inducibletargeting of IL-13 to the adult lung causes matrix metalloproteinase-andcathepsin-dependent emphysema. J Clin Invest 106:1081-1093). Recentworks describe involvement of septal cell apoptosis in lung tissuedestruction leading to emphysema (Rangasami T, et al. Genetic ablationof Nrf2 enhances susceptibility to cigarette smoke-iduced emphysema inmice. Submitted to Journal of Clinincal Investigation; Tuder R M et al.Oxidative stress and apoptosis interact and cause emphysema due tovascular endothelial growth factor blocade. Am J Respir Cell Mol Biol,29:88-97; 2003.; Yokohori N, Aoshiba K, Nagai A, Increased levels ofcell death and proliferation in alveolar wall cells in patients withpulmonary emphysema. Chest. 2004 February; 125(2):626-32.; Aoshiba K,Yokohori N, Nagai A., Alveolar wall apoptosis causes lung destructionand emphysematous changes. Am J Respir Cell Mol. Biol. 2003 May;28(5):555-62.).

Among the mechanisms that underlie both pathways of lung destruction inemphysema, excessive formation of reactive oxygen species (ROS) shouldbe first of all mentioned. It is well established thatprooxidant/antioxidant imbalance exists in the blood and in the lungtissue of smokers (Hulea S A, et al: Cigarette smoking causesbiochemical changes in blood that are suggestive of oxidative stress: acase-control study. J Environ Pathol Toxicol Oncol. 1995;14(3-4):173-80.; Rahman I, MacNee W. Lung glutathione and oxidativestress: implications in cigarette smoke-induced airway disease. Am J.Physiol. 1999 December; 277(6 Pt 1):L1067-88.; MacNee W.Oxidants/antioxidants and COPD. Chest. 2000 May; 117(5 Suppl1):303S-17S.; Marwick J A, Kirkham P, Gilmour P S, Donaldson K, MacNEEW, Rahman I. Cigarette smoke-induced oxidative stress and TGF-beta1increase p21waf1/cip1 expression in alveolar epithelial cells. Ann N YAcad Sci. 2002 November; 973:278-83.; Aoshiba K, Koinuma M, Yokohori N,Nagai A. Immunohistochemical evaluation of oxidative stress in murinelungs after cigarette smoke exposure. Inhal Toxicol. 2003 September;15(10):1029-38.; Dekhuijzen P N. Antioxidant properties ofN-acetylcysteine: their relevance in relation to chronic obstructivepulmonary disease. Eur Respir J. 2004 April; 23(4):629-36.; Tuder R M,Zhen L, Cho C Y, Taraseviciene-Stewart L, Kasahara Y, Salvemini D,Voelkel N F, and Flores S C. Oxidative stress and apoptosis interact andcause emphysema due to vascular endothelial growth factor blocade. Am JRespir Cell Mol Biol, 29:88-97; 2003.). After one hour exposure of miceto CS, there is a dramatic increase of 8-hydroxy-2′-deoxyguanosine(8-OHdG) in the alveolar epithelial cells, particularly of type II (seeInhal Toxicol. 2003 September; 15(10):1029-38. above).

Overproduced reactive oxygen species are known for their cytotoxicactivity, which stems from a direct DNA damaging effect and from theactivation of apoptotic signal transduction pathways (Takahashi A,Masuda A, Sun M, Centonze V E, Herman B. Oxidative stress-inducedapoptosis is associated with alterations in mitochondrial caspaseactivity and Bcl-2-dependent alterations in mitochondrial pH (pHm).Brain Res Bull. 2004 Feb. 15; 62(6):497-504.; Taniyama Y, Griendling KK. Reactive oxygen species in the vasculature: molecular and cellularmechanisms. Hypertension. 2003 December; 42(6):1075-81. Epub 2003 Oct.27.; Higuchi Y. Chromosomal DNA fragmentation in apoptosis and necrosisinduced by oxidative stress. Biochem Pharmacol. 2003 Oct. 15;66(8):1527-35.; Punj V, Chakrabarty A M. Redox proteins in mammaliancell death: an evolutionarily conserved function in mitochondria andprokaryotes. Cell Microbiol. 2003 April; 5(4):225-31.; Ueda S, MasutaniH, Nakamura H, Tanaka T, Ueno M, Yodoi J. Redox control of cell death.Antioxid Redox Signal. 2002 June; 4(3):405-14.).

ROS's are not only cytotoxic per se but are also proinflammatorystimuli, being prominent activators of redox-sensitive transcriptionfactors NFkB and AP-1 (reviewed in Rahman I. Oxidative stress and genetranscription in asthma and chronic obstructive pulmonary disease:antioxidant therapeutic targets. Curr Drug Targets Inflamm Allergy. 2002September; 1(3):291-315.). Both transcription factors are, in turn,strongly implicated in stimulation of transcription of proinflammatorycytokines (reviewed in Renard P, Raes M. The proinflammatorytranscription factor NFkappaB: a potential target for noveltherapeutical strategies. Cell Biol Toxicol. 1999; 15(6):341-4.; LentschA B, Ward Pa. The NFkappaBb/IkappaB system in acute inflammation. ArchImmunol Ther Exp (Warsz). 2000; 48(2):59-63) and matrix degradingproteinases (Andela V B, Gordon A H, Zotalis G, Rosier R N, Goater J J,Lewis G D, Schwarz E M, Puzas J E, O'Keefe R J. NFkappaB: a pivotaltranscription factor in prostate cancer metastasis to bone. Clin Orthop.2003 October; (415 Suppl):S75-85.; Fleenor D L, Pang I H, Clark A F.Involvement of AP-1 in interleukin-1alpha-stimulated MMP-3 expression inhuman trabecular meshwork cells. Invest Ophthalmol Vis Sci. 2003 August;44(8):3494-501.; Ruhul Amin A R, Senga T, Oo M L, Thant A A, HamaguchiM. Secretion of matrix metalloproteinase-9 by the proinflammatorycytokine, IL-1beta: a role for the dual signalling pathways, Akt andErk. Genes Cells. 2003 June; 8(6):515-23.). Proinflammatory cytokines,in turn, serve as attractors of inflammatory cells that also secretematrix degrading enzymes, cytokines and reactive oxygen species. Thus,it appears that a pathogenic factor, like e.g. CS, triggers apathological network where reactive oxygen species act as majormediators of lung destruction.

Both reactive oxygen species (ROS) from inhaled cigarette smoke andthose endogenously formed by inflammatory cells contribute to anincreased intrapulmonary oxidant burden.

One additional pathogenic factor with regards to COPD pathogenesis isthe observed decreased expression of VEGF and VEGFRII in lungs ofemphysematous patients (Yasunori Kasahara, Rubin M. Tuder, Carlyne D.Cool, David A. Lynch, Sonia C. Flores, and Norbert F. Voelkel.Endothelial Cell Death and Decreased Expression of Vascular EndothelialGrowth Factor and Vascular Endothelial Growth Factor Receptor 2 inEmphysema. Am J Respir Crit Care Med Vol 163. pp 737-744, 2001).Moreover, inhibition of VEGF signaling using chemical VEGFR inhibitorleads to alveolar septal endothelial and then to epithelial cellapoptosis, probably due to disruption of intimate structural/functionalconnection of both types of cells within alveoli (Yasunori Kasahara,Rubin M. Tuder, Laimute Taraseviciene-Stewart, Timothy D. Le Cras,Steven Abman, Peter K. Hirth, Johannes Waltenberger, and Norbert F.Voelkel. Inhibition of VEGF receptors causes lung cell apoptosis andemphysema. J. Clin. Invest. 106:1311-1319 (2000).; Voelkel N F, Cool CD. Pulmonary vascular involvement in chronic obstructive pulmonarydisease. Eur Respir J Suppl. 2003 November; 46:28s-32s).

Macular Degeneration

The most common cause of decreased best-corrected vision in individualsover 65 years of age in the US is the retinal disorder known asage-related macular degeneration (AMD). As AMD progresses, the diseaseis characterized by loss of sharp, central vision. The area of the eyeaffected by AMD is the Macula—a small area in the center of the retina,composed primarily of photoreceptor cells. So-called “dry” AMD,accounting for about 85%-90% of AMD patients, involves alterations ineye pigment distribution, loss of photoreceptors and diminished retinalfunction due to overall atrophy of cells. So-called “wet” AMD involvesproliferation of abnormal choroidal vessels leading to clots or scars inthe sub-retinal space. Thus, the onset of wet AMD occurs because of theformation of an abnormal choroidal neovascular network (choroidalneovascularization, CNV) beneath the neural retina. The newly formedblood vessels are excessively leaky. This leads to accumulation ofsubretinal fluid and blood leading to loss of visual acuity. Eventually,there is total loss of functional retina in the involved region, as alarge disciform scar involving choroids and retina forms. While dry AMDpatients may retain vision of decreased quality, wet AMD often resultsin blindness. (Hamdi & Kenney, Age-related Macular degeneration—a newviewpoint, Frontiers in Bioscience, e305-314, May 2003). CNV occurs notonly in wet AMD but also in other ocular pathologies such as ocularhistoplasmosis syndrome, angiod streaks, ruptures in Bruch's membrane,myopic degeneration, ocular tumors and some retinal degenerativediseases.

Various studies conducted have determined several risk factors for AMD,such as smoking, aging, family history (Milton, Am J Ophthalmol 88, 269(1979); Mitchell et al., Ophthalmology 102, 1450-1460 (1995); Smith etal., Ophthalmology 108, 697-704 (2001)) sex (7-fold higher likelihood infemales: Klein et al., Ophthalmology 99, 933-943 (1992) and race (whitesare most susceptible). Additional risk factors may include eyecharacteristics such as farsightedness (hyperopia) and light-coloredeyes, as well as cardiovascular disease and hypertension. Evidence ofgenetic involvement in the onset progression of the disease has alsobeen documented (see Hamdi & Kenney above).

Two companies, Acuity Pharmaceuticals and Sima Therapeutics, have bothrecently filed an IND for siRNA molecules inhibiting VEGF and VEGF-R1(Flt-1), respectively, for treatment of AMD. These molecules are termedCand5 inhibitor and 027 inhibitor respectively.

Microvascular Disorders

Microvascular disorders are composed of a broad group of conditions thatprimarily affect the microscopic capillaries and lymphatics and aretherefore outside the scope of direct surgical intervention.Microvascular disease can be broadly grouped into the vasospastic, thevasculitis and lymphatic occlusive. Additionally, many of the knownvascular conditions have a microvascular element to them.

-   -   Vasospastic Disease—Vasospastic diseases are a group of        relatively common conditions where, for unknown reasons, the        peripheral vasoconstrictive reflexes are hypersensitive. This        results in inappropriate vasoconstriction and tissue ischaemia,        even to the point of tissue loss. Vasospastic symptoms are        usually related to temperature or the use of vibrating machinery        but may be secondary to other conditions.    -   Vasculitic Disease—Vasculitic diseases are those that involve a        primary inflammatory process in the microcirculation. Vasculitis        is usually a component of an autoimmune or connective tissue        disorder and is not generally amenable to surgical treatment but        requires immunosuppressive treatment if the symptoms are severe.    -   Lymphatic Occlusive Disease—Chronic swelling of the lower or        upper limb (lymphoedema) is the result of peripheral lymphatic        occlusion. This is a relatively rare condition that has a large        number of causes, some inherited, some acquired. The mainstays        of treatment are correctly fitted compression garments and the        use of intermittent compression devices.        Microvascular Pathologies Associated with Diabetes

Diabetes is the leading cause of blindness, the number one cause ofamputations and impotence, and one of the most frequently occurringchronic childhood diseases. Diabetes is also the leading cause ofend-stage renal disease in the United States, with a prevalence rate of31% compared with other renal diseases. Diabetes is also the mostfrequent indication for kidney transplantation, accounting for 22% ofall transplantation operations.

In general, diabetic complications can be classified broadly asmicrovascular or macrovascular disease. Microvascular complicationsinclude neuropathy (nerve damage), nephropathy (kidney disease) andvision disorders (eg retinopathy, glaucoma, cataract and cornealdisease). In the retina, glomerulus, and vasa nervorum, similarpathophysiologic features characterize diabetes-specific microvasculardisease.

Microvascular pathologies associated with diabetes are defined as adisease of the smallest blood vessels (capillaries) that may occur e.g.in people who have had diabetes for a long time. The walls of thevessels become abnormally thick but weak. They, therefore, bleed, leakprotein and slow the flow of blood through the body.

Clinical and animal model data indicate that chronic hyperglycemia isthe central initiating factor for all types of diabetic microvasculardisease. Duration and magnitude of hyperglycemia are both stronglycorrelated with the extent and rate of progression of diabeticmicrovascular disease. Although all diabetic cells are exposed toelevated levels of plasma glucose, hyperglycemic damage is limited tothose cell types (e.g., endothelial cells) that develop intracellularhyperglycemia. Endothelial cells develop intracellular hyperglycemiabecause, unlike many other cells, they cannot down-regulate glucosetransport when exposed to extracellular hyperglycemia. Thatintracellular hyperglycemia is necessary and sufficient for thedevelopment of diabetic pathology is further demonstrated by the factthat overexpression of the GLUT1 glucose transporter in mesangial cellscultured in a normal glucose milieu mimics the diabetic phenotype,inducing the same increases in collagen type IV, collagen type I, andfibronectin gene expression as diabetic hyperglycemia.

Abnormal Endothelial Cell Function: Early in the course of diabetesmellitus, before structural changes are evident, hyperglycemia causesabnormalities in blood flow and vascular permeability in the retina,glomerulus, and peripheral nerve vasa nervorum. The increase in bloodflow and intracapillary pressure is thought to reflecthyperglycemia-induced decreased nitric oxide (NO) production on theefferent side of capillary beds, and possibly an increased sensitivityto angiotensin II. As a consequence of increased intracapillary pressureand endothelial cell dysfunction, retinal capillaries exhibit increasedleakage of fluorescein and glomerular capillaries have an elevatedalbumin excretion rate (AER). Comparable changes occur in the vasavasorum of peripheral nerve. Early in the course of diabetes, increasedpermeability is reversible; as time progresses, however, it becomesirreversible.

Increased Vessel Wall Protein Accumulation

The common pathophysiologic feature of diabetic microvascular disease isprogressive narrowing and eventual occlusion of vascular lumina, whichresults in inadequate perfusion and function of the affected tissues.Early hyperglycemia-induced microvascular hypertension and increasedvascular permeability contribute to irreversible microvessel occlusionby three processes:

-   -   The first is an abnormal leakage of periodic acid-Schiff        (PAS)-positive, carbohydrate-containing plasma proteins, which        are deposited in the capillary wall and which may stimulate        perivascular cells such as pericytes and mesangial cells to        elaborate growth factors and extracellular matrix.    -   The second is extravasation of growth factors, such as        transforming growth factor β1 (TGF-β1), which directly        stimulates overproduction of extracellular matrix components,        and may induce apoptosis in certain complication-relevant cell        types.    -   The third is hypertension-induced stimulation of pathologic gene        expression by endothelial cells and supporting cells, which        include glut-1 glucose transporters, growth factors, growth        factor receptors, extracellular matrix components, and adhesion        molecules that can activate circulating leukocytes. The        observation that unilateral reduction in the severity of        diabetic microvascular disease occurs on the side with        ophthalmic or renal artery stenosis is consistent with this        concept.        Microvascular Cell Loss and Vessel Occlusion

The progressive narrowing and occlusion of diabetic microvascular luminaare also accompanied by microvascular cell loss. In the retina, diabetesmellitus induces programmed cell death of Müller cells and ganglioncells, pericytes, and endothelial cells. In the glomerulus, decliningrenal function is associated with widespread capillary occlusion andpodocyte loss, but the mechanisms underlying glomerular cell loss arenot yet known. In the vasa nervorum, endothelial cell and pericytedegeneration occur, and these microvascular changes appear to precedethe development of diabetic peripheral neuropathy. The multifocaldistribution of axonal degeneration in diabetes supports a causal rolefor microvascular occlusion, but hyperglycemia-induced decreases inneurotrophins may contribute by preventing normal axonal repair andregeneration.

Another common feature of diabetic microvascular disease has been termedhyperglycemic memory, or the persistence or progression ofhyperglycemia-induced microvascular alterations during subsequentperiods of normal glucose homeostasis. The most striking example of thisphenomenon is the development of severe retinopathy in histologicallynormal eyes of diabetic dogs that occurred entirely during a 2.5-yearperiod of normalized blood glucose that followed 2.5 years ofhyperglycemia. Hyperglycemia-induced increases in selected matrix genetranscription also persist for weeks after restoration of normoglycemiain vivo, and a less pronounced, but qualitatively similar, prolongationof hyperglycemia-induced increase in selected matrix gene transcriptionoccurs in cultured endothelial cells.

For further information, see “Shared pathophysiologic features ofmicrovascular complications of diabetes” (Larsen: Williams Textbook ofEndocrinology, 10th ed., Copyright © 2003 Elsevier).

Microvascular complications occur not only in overt diabetes but arealso due to Impaired Glucose Tolerance (IGT). Microvascularcomplications of IGT: neuropathy, retinopathy, and renalmicroproteinuria.

Diabetic Neuropathy

Diabetic neuropathies are neuropathic disorders (peripheral nervedamage) that are associated with diabetes mellitus. These conditionsusually result from diabetic microvascular injury involving small bloodvessels that supply nerves (vasa nervorum). Relatively common conditionswhich may be associated with diabetic neuropathy include third nervepalsy; mononeuropathy; mononeuropathy multiplex; diabetic amyotrophy; apainful polyneuropathy; autonomic neuropathy; and thoracoabdominalneuropathy and the most common form, peripheral neuropathy, which mainlyaffects the feet and legs. There are four factors involved in thedevelopment of diabetic neuropathy: microvascular disease, advancedglycated end products, protein kinase C, and the polyol pathway.

Microvascular Disease in Diabetic Neuropathy

Vascular and neural diseases are closely related and intertwined. Bloodvessels depend on normal nerve function, and nerves depends on adequateblood flow. The first pathological change in the microvasculature isvasoconstriction. As the disease progresses, neuronal dysfunctioncorrelates closely with the development of vascular abnormalities, suchas capillary basement membrane thickening and endothelial hyperplasia,which contribute to diminished oxygen tension and hypoxia. Neuronalischemia is a well-established characteristic of diabetic neuropathy.Vasodilator agents (e.g., angiotensin-converting-enzyme inhibitors,alpha1-antagonists) can lead to substantial improvements in neuronalblood flow, with corresponding improvements in nerve conductionvelocities. Thus, microvascular dysfunction occurs early in diabetes,parallels the progression of neural dysfunction, and may be sufficientto support the severity of structural, functional, and clinical changesobserved in diabetic neuropathy. Peripheral neuropathy (legs),sensorimotor neuropathy is a significant component in the pathogenesisof leg ulcers in diabetes.

Neuropathy is a common complication of diabetes occurring over time inmore than half of patients with type 2 diabetes. Nerve conductionstudies demonstrate that neuropathy is already present in 10-18% ofpatients at the time of diabetes diagnosis, suggesting that peripheralnerve injury occurs at early stages of disease and with milder glycemicdysregulation. The concept that neuropathy is an early clinical sign ofdiabetes was proposed >40 years ago, and most studies report anassociation between IGT and neuropathy. Most patients with IGT andassociated neuropathy have a symmetric, distal sensory polyneuropathywith prominent neuropathic pain. IGT neuropathy (Microvascularcomplications of impaired glucose tolerance—Perspectives in Diabetes, J.Robinson Singleton, in Diabetes Dec. 1, 2003) is phenotypically similarto early diabetic neuropathy, which also causes sensory symptoms,including pain, and autonomic dysfunction. In a survey of 669 patientswith early diabetic neuropathy, sensory symptoms were present in >60%,impotence in nearly 40%, and other autonomic involvement in 33%, butevidence of motor involvement in only 12%. These clinical findingssuggest prominent early involvement of the small unmyelinated nervefibers that carry pain, temperature, and autonomic signals. Directquantitation of unmyelinated intraepidermal nerve fibers from skinbiopsies shows similar fiber loss and altered morphology in patientswith neuropathy associated with IGT and early diabetes.

Autonomic dysfunction, particularly erectile dysfunction and alteredcardiac vagal response, are common early features of neuropathic injuryin diabetes. Work with IGT patients also suggests prevalent vagaldysautonoinia: separate studies have found abnormal heart rate recoveryfollowing exercise, blunted R—R interval variability to deep breathing,and reduced expiration to inspiration ratio (all measures of vagaldysautonomia) in a greater fraction of IGT patients than age-matchednormoglycemic control subjects.

Nerve damage in diabetes affects the motor, sensory, and autonomicfibers. Motor neuropathy causes muscle weakness, atrophy, and paresis.Sensory neuropathy leads to loss of the protective sensations of pain,pressure, and heat. The absence of pain leads to many problems in theinsensate foot, including ulceration, unperceived trauma, and Charcotneuroarthropathy. The patient may not seek treatment until after thewound has advanced. A combination of sensory and motor dysfunction cancause the patient to place abnormal stresses on the foot, resulting intrauma, which may lead to infection. Autonomic sympathetic neuropathycauses vasodilation and decreased sweating, which results in warm,overly dry feet that are particularly prone to skin breakdown, as wellas functional alterations in microvascular flow. Autonomic dysfunction(and denervation of dermal structures) also results in loss of skinintegrity, which provides an ideal site for microbial invasion. Theneuropathic foot does not ulcerate spontaneously; rather, it is thecombination of some form of trauma accompanied by neuropathy.

Microvascular dysfunction occurs early in diabetes, parallels theprogression of neural dysfunction, and may be sufficient to support theseverity of structural, functional, and clinical changes observed indiabetic neuropathy.

Advanced glycated end products—Elevated intracellular levels of glucosecause a non-enzymatic covalent bonding with proteins, which alters theirstructure and destroys their function. Certain of these glycatedproteins are implicated in the pathology of diabetic neuropathy andother long term complications of diabetes.

Protein kinase C (PKC)—PKC is implicated in the pathology of diabeticneuropathy. Increased levels of glucose cause an increase inintracellular diacylglycerol, which activates PKC. PKC inhibitors inanimal models will increase nerve conduction velocity by increasingneuronal blood flow.

Sensorimotor Polyneuropathy

Longer nerve fibers are affected to a greater degree than shorter ones,because nerve conduction velocity is slowed in proportion to a nerve'slength. In this syndrome, decreased sensation and loss of reflexesoccurs first in the toes bilaterally, then extends upward. It is usuallydescribed as glove-stocking distribution of numbness, sensory loss,dysesthesia and nighttime pain. The pain can feel like burning, prickingsensation, achy or dull. Pins and needles sensation is common. Loss ofproprioception, that is, the sense of where a limb is in space, isaffected early. These patients cannot feel when they are stepping on aforeign body, like a splinter, or when they are developing a callousfrom an ill-fitting shoe. Consequently, they are at risk for developingulcers and infections on the feet and legs, which can lead toamputation. Similarly, these patients can get multiple fractures of theknee, ankle or foot, and develop a Charcot joint. Loss of motor functionresults on dorsiflexion contractures of the toes, so called hammertoes.These contractures occur not only in the foot but also in the hand.

Autonomic Neuropathy

The autonomic nervous system is composed of nerves serving the heart, GItract and urinary system. Autonomic neuropathy can affect any of theseorgan systems. The most commonly recognized autonomic dysfuction indiabetics is orthostatic hypotension, or the uncomfortable sensation offainting when a patient stands up. In the case of diabetic autonomicneuropathy, it is due to the failure of the heart and arteries toappropriately adjust heart rate and vascular tone to keep bloodcontinually and fully flowing to the brain. This symptom is usuallyaccompanied by a loss of sinus respiratory variation, that is, the usualchange in heart rate seen with normal breathing. When these two findingsare present, cardiac autonomic neuropathy is present.

GI tract manifestations include delayed gastric emptying, gastroparesis,nausea, bloating, and diarrhea. Because many diabetics take oralmedication for their diabetes, absorption of these medicines is greatlyaffected by the delayed gastric emptying. This can lead to hypoglycemiawhen an oral diabetic agent is taken before a meal and does not getabsorbed until hours, or sometimes days later, when there is normal orlow blood sugar already. Sluggish movement of the small instestine cancause bacterial overgrowth, made worse by the presence of hyperglycemia.This leads to bloating, gas and diarrhea.

Urinary symptoms include urinary frequency, urgency, incontinence andretention. Again, because of the retention of sweet urine, urinary tractinfections are frequent. Urinary retention can lead to bladderdiverticula, stones, reflux nephropathy.

Cranial Neuropathy

When cranial nerves are affected, oculomotor (3rd) neuropathies are mostcommon. The oculomotor nerve controls all of the muscles that move theeye with the exception of the lateral rectus and superior obliquemuscles. It also serves to constrict the pupil and open the eyelid. Theonset of a diabetic third nerve palsy is usually abrupt, beginning withfrontal or periorbital pain and then diplopia. All of the oculomotormuscles innervated by the third nerve may be affected, except for thosethat control pupil size. The sixth nerve, the abducens nerve, whichinnervates the lateral rectus muscle of the eye (moves the eyelaterally), is also commonly affected but fourth nerve, the trochlearnerve, (innervates the superior oblique muscle, which moves the eyedownward) involvement is unusual. Mononeuropathies of the thoracic orlumbar spinal nerves can occur and lead to painful syndromes that mimicmyocardial infarction, cholecystitis or appendicitis. Diabetics have ahigher incidence of entrapment neuropathies, such as carpal tunnelsyndrome.

Diabetic Limb Ischemia and Diabetic Foot Ulcers

Diabetes and pressure can impair microvascular circulation and lead tochanges in the skin on the lower extremities, which in turn, can lead toformation of ulcers and subsequent infection. Microvascular changes leadto limb muscle microangiopathy, as well as a predisposition to developperipheral ischemia and a reduced angiogenesis compensatory response toischemic events. Microvascular pathology exacerbates Peripheral VascularDisease (PVD) (or Peripheral Arterial Disease (PAD) or Lower ExtremityArterial Disease (LEAD)—a MACROvascular complication—narrowing of thearteries in the legs due to atherosclerosis. PVD occurs earlier indiabetics, is more severe and widespread, and often involvesintercurrent microcirculatory problems affecting the legs, eyes, andkidneys.

Foot ulcers and gangrene are frequent comorbid conditions of PAD.Concurrent peripheral neuropathy with impaired sensation make the footsusceptible to trauma, ulceration, and infection. The progression of PADin diabetes is compounded by such comorbidity as peripheral neuropathyand insensitivity of the feet and lower extremities to pain and trauma.With impaired circulation and impaired sensation, ulceration andinfection occur. Progression to osteomyelitis and gangrene maynecessitate amputation.

Persons with diabetes are up to 25 times more likely than nondiabeticpersons to sustain a lower limb amputation, underscoring the need toprevent foot ulcers and subsequent limb loss. Diabetic foot ulcers mayoccur not only in conjunction with PAD but may also be associated withneuropathy, venous insufficiency (varicose veins), trauma, andinfection. PAD contributes to these other conditions in producing orprecipitating foot ulcers. Foot ulcers do not necessarily representprogression of PAD, as they may occur in the presence of adequateclinical peripheral arterial perfusion. Patient-based studies indicatean increased risk of foot ulceration in diabetic patients who haveperipheral neuropathy and a high plantar foot pressure. The prevalenceof a history of ulcers or sores on the foot or ankles was 15% of alldiabetic patients in the population-based study in southern Wisconsin.The prevalence was higher for diabetic individuals diagnosed at age <30years, was slightly higher in men (16%) than in women (13%), and wasgreater in insulin-treated diabetic patients (17%) than in patients nottaking insulin (10%). The prevalence increased with age, especially indiabetic patients diagnosed at age <30 years. In patient studies fromEurope, prevalence of foot ulcers in diabetic patients was 3% in thoseage <50 years, 7% in those age <60 years, and 14% in those age <80years. Prevalence was greater in males than in females at age 70 years.

In diabetic patients, foot ischemia and infection are serious and evenlife-threatening occurrences; however, neuropathy is the most difficultcondition to treat. The medical and surgical literature concerning allaspects of the clinical and pathological manifestations of the diabeticfoot is overwhelming. Neuropathy, angiopathy, retinopathy, andnephropathy, alone or in combination and in varying degrees of severity,may influence the treatment of the diabetic foot.

Every year, 82,000 limb amputations are performed in patients withdiabetes mellitus. The majority of these amputations are performed inthe elderly population. Amputations resulting from diabetes may arisefrom multiple etiologies, including foot ulcers, ischemia, venous legulcers (ie, those secondary to venous reflux), and heel ulcers (ie,those resulting from untreated pressure ulcers in the heel). Themajority of these amputations originate from ulcers. The prevalence offoot ulcers among patients with diabetes is 12%. In addition, the20-year cumulative incidence of lower-extremity ulcers in patients withtype 1 diabetes is 9.9%. Diabetes-induced limb amputations result in a5-year mortality rate of 39% to 68% and are associated with an increasedrisk of additional amputations. The length of hospital stay isapproximately 60% longer among patients with diabetic foot ulcers, ascompared with those without ulcers.

Diabetic neuropathy impairs the nerve axon reflex that depends onhealthy C-fiber nociceptor function and causes local vasodilation inresponse to a painful stimulus. This condition further compromises thevasodilatory response present in conditions of stress, such as injury orinflammation, in the diabetic neuropathic foot. This impairment maypartially explain why some ulcers in the diabetic neuropathic foot areeither slow to heal or fail to heal at all, despite successfullower-extremity revascularization.

The most common causal pathway to diabetic foot ulceration can thus beidentified as the combination of neuropathy (sensory loss), deformity(eg, prominent metatarsal heads), and trauma (eg, ill-fitting footwear).

Most surgeons prefer to perform popliteal or tibial arterial bypassbecause of inferior rates of limb salvage and patency compared with moreproximal procedures. If popliteal or tibial arterial bypass is unable torestore a palpable foot pulse, pedal bypass has been reported to providea more durable and effective limb-salvage procedure for patients withdiabetes and ischemic foot wounds]. Even extensive multisegmentocclusive disease in patients with diabetes does not present animpediment to foot salvage. Whereas serious wound complications may havedisastrous results, they are uncommon after pedal bypass grafting.Adequate control of preexisting foot infection and careful grafttunneling have been shown to be effective in avoiding furthercomplications. Angioplasty in the lower extremity is becoming moreprogressively utilized. However, it must be emphasized that forangioplasty to be effective, a distal vessel or feeding vessel must bepatent if the more proximal angioplasty is to succeed.

While diabetic ulcers/limb pathologies may be managed in some patients(by Debridement, antibiotic treatment, use of preparations to stimulategranulation tissue (new collagen and angiogenesis) and reduction ofbacterial burden in the wound), it would be beneficial to have apharmaceutical composition that could better treat these conditionsand/or alleviate the symptoms.

For further information, see American Journal of Surgery, Volume187•Number 5 Suppl 1•May 1, 2004, Copyright © 2004 Elsevier.

Coronary Microvascular Dysfunction in Diabetes

The correlation between histopathology and microcirculatory dysfunctionin diabetes is well known from old experimental studies and fromautopsy, where thickening of the basal membrane, perivascular fibrosis,vascular rarefication, and capillary hemorrhage are frequently found. Itremains difficult to confirm these data in vivo, although a recent paperdemonstrated a correlation between pathology and ocular micorovasculardysfunction (Am J Physiol 2003; 285). A large amount of clinicalstudies, however, indicate that not only overt diabetes but alsoimpaired metabolic control may affect coronary microcirculation (HypertRes 2002; 25:893). Werner alluded to the important paper by Sambuceti etal (Circulation 2001; 104:1129) showing the persistence of microvasculardysfunction in patients after successful reopening of the infarctrelated artery, and which may explain the increased cardiovascularmorbidity and mortality in these patients. There is mounting evidencefrom large acute reperfusion studies that morbidity and mortality areunrelated to the reopening itself of the infarct related artery, butmuch more dependent on the TIMI flow+/− myocardial blush (Stone 2002;Feldmann Circulation 2003). Herrmann indicated, among others, that theintegrity of the coronary microcirculation is probably the mostimportant clincal and prognostic factor in this context (Circulation2001). The neutral effect of protection devices (no relevant change forTIMI flow, for ST resolution, or for MACE) may indicate that afunctional impairment of microcirculation is the major determinant ofprognosis. There is also increasing evidence that coronary microvasculardysfunction plays a major role in non obstructive CAD. Coronaryendothelial dysfunction remains a strong prognostic predictor in thesepatients.

Diabetic Nephropathy (Renal Dysfunction in Patients with Diabetes)

Diabetic nephropathy encompasses microalbuminuria (a microvasculardisease effect), proteinuria and ESRD. Diabetes is the most common causeof kidney failure, accounting for more than 40 percent of new cases.Even when drugs and diet are able to control diabetes, the disease canlead to nephropathy and kidney failure. Most people with diabetes do notdevelop nephropathy that is severe enough to cause kidney failure. About16 million people in the United States have diabetes, and about 100,000people have kidney failure as a result of diabetes.

Diabetic Retinopathy

In the diabetic state, hyperglycemia leads to decreased retinal bloodflow, retinal hyperpermeability, delays in photoreceptor nerveconduction, and retinal neuronal cell death. In short duration diabetes,neuronal cell death has been identified within the inner nuclear layerof the retina. Specifically, apoptosis has been localized to glial cellssuch as Mueller cells and astrocytes and has been shown to occur within1 month of diabetes in the STZ-induced diabetic rat model. The cause ofthese events is multi-factorial including activation of thediacylglycerol/PKC pathway, oxidative stress, and nonenzymaticglycosylation. The combination of these events renders the retinahypoxic and ultimately leads to the development of diabetic retinopathy.One possible connection between retinal ischemia and the early changesin the diabetic retina is the hypoxia-induced production of growthfactors such as VEGF. The master regulator of the hypoxic response hasbeen identified as hypoxia inducible factor-1 (HIF-1), which controlsgenes that regulate cellular proliferation and angiogenesis. Priorstudies have demonstrated that inhibition of HIF-1 ubiquitination leadsto binding with hypoxia responsive elements (HRE) and production of VEGFmRNA.

Diabetic Retinopathy is defined as the progressive dysfunction of theretinal vasculature caused by chronic hyperglycemia. Key features ofdiabetic retinopathy include microaneurysms, retinal hemorrhages,retinal lipid exudates, cotton-wool spots, capillary nonperfusion,macular edema and neovascularization. Associated features includevitreous hemorrhage, retinal detachment, neovascular glaucoma, prematurecataract and cranial nerve palsies.

There are 16 million people in the US with Type 1 and Type 2 diabetes.Within 15 years, 80% of Type 1 patients have developed diabeticretinopathy while 84% of Type 2 diabetic patients develop retinopathywithin 19 years. These numbers constitute a significant market fortherapeutic agents aimed at ocular diseases of neovasculature. Thedevelopment of diabetic retinopathy is time-dependent. Despite optimalblood sugar control, patients with long-standing disease can be expectedto eventually develop some form of retinopathy. The National Society toPrevent Blindness has estimated that 4 to 6 million diabetics in theU.S. have diabetic retinopathy. The estimated annual incidence of newcases of proliferative diabetic retinopathy and diabetic macular edemaare 65,000 and 75,000, respectively, with a prevalence of 700,000 and500,000 respectively. Diabetic retinopathy causes from 12,000 to 24,000new cases of blindness in the US every year. Retinopathy is treated bysurgical methods, effective in reducing severe vision loss, but thelasered portions of the retina are irreversibly destroyed. There are nodrug treatments available.

A microvascular disease that primarily affects the capillaries, diabetesmellitus affects the eye by destroying the vasculature in theconjunctiva, retina and central nervous system. Patients may presentwith histories of long-standing injected bulbar conjunctivae along withsystemic complaints of weight loss despite larger than normal appetite(polyphasia), abnormal thirst (polydypsia) and abnormally frequenturination (polyuria).

Fluctuating visual acuity secondary to unstable blood sugar is a commonocular sign. Swelling within the crystalline lens results in largesudden shifts in refraction as well as premature cataract formation.Changes in visual acuity will depend upon the severity and stage of thedisease.

In the retina, weakening of the arterioles and capillaries may result inthe characteristic appearance of intraretinal dot and blot hemorrhages,exudates, intraretinal microvascular abnormalities (IRMA)microaneurysms, edema and cotton wool infarcts. Proliferative diabeticretinopathy is the result of severe vascular compromise and is visibleas neovascularization of the disc (NVD), neovascularization elsewhere(NVE) and neovascularization of the iris (NVI, or rubeosis irides).Neurological complications include palsies of the third, fourth andsixth cranial nerves as well as diabetic papillitis and facial nerveparalysis.

Diabetes mellitus is a genetically influenced group of diseases thatshare glucose intolerance. It is characterized as a disorder ofmetabolic regulation as a result of deficient or malfunctioning insulinor deficient or malfunctioning cellular insulin receptors.

Biochemistry involving the formation of sorbitol plays a role in thedestruction of pericytes, which are cells that support the vascularendothelium. As the supportive pericytes perish, capillary endotheliumbecomes compromised, resulting in the vascular leakage of blood, proteinand lipid. This, in combination with thickened, glucose-laden blood,produces vascular insufficiency, capillary nonperfusion, retinalhypoxia, altered structure and decreased function. The formation andrelease of vasoproliferative factors which play a role in the genesis ofretinal neovascularization are poorly understood.

Most non-vision threatening sequelae of diabetes resolve spontaneouslyover the course of weeks to months following medical control. In caseswhere there are large refractive changes, patients may require atemporary spectacle prescription until the refraction stabilizes. Whenretinopathy threatens the macula or when new blood vessels proliferate,the patient may be referred for laser photocoagulation. The DiabeticRetinopathy Study (DRS) has conclusively proven that panretinalphotocoagulation was successful in reducing the risk of severe visionloss in high-risk patients. It defined the high-risk characteristics as:(1) Neovascularization of the optic disc (NVD) one-quarter to one-thirdof a disc diameter in size and (2) Neovascularization elsewhere (NVE)with any vitreous hemorrhage.

Diabetic Macular Edema (DME)

DME is a complication of diabetic retinopathy, a disease affecting theblood vessels of the retina. Diabetic retinopathy results in multipleabnormalities in the retina, including retinal thickening and edema,hemorrhages, impeded blood flow, excessive leakage of fluid from bloodvessels and, in the final stages, abnormal blood vessel growth. Thisblood vessel growth can lead to large hemorrhages and severe retinaldamage. When the blood vessel leakage of diabetic retinopathy causesswelling in the macula, it is referred to as DME. The principal symptomof DME is a loss of central vision. Risk factors associated with DMEinclude poorly controlled blood glucose levels, high blood pressure,abnormal kidney function causing fluid retention, high cholesterollevels and other general systemic factors.

According to the World Health Organization, diabetic retinopathy is theleading cause of blindness in working age adults and a leading cause ofvision loss in diabetics. The American Diabetes Association reports thatthere are approximately 18 million diabetics in the United States andapproximately 1.3 million newly diagnosed cases of diabetes in theUnited States each year. Prevent Blindness America and the National EyeInstitute estimate that in the United States there are over 5.3 millionpeople aged 18 or older with diabetic retinopathy, includingapproximately 500,000 with DME. The CDC estimates that there areapproximately 75,000 new cases of DME in the United States each year.

Additional Neuropathies

In addition to diabetes, the common causes of neuropathy are herpeszoster infection, chronic or acute trauma (including surgery) andvarious neurotoxins. Neuropathic pain is common in cancer as a directresult of the cancer on peripheral nerves (e.g., compression by a tumor)and as a side effect of many chemotherapy drugs.

Microvascular disease—Vascular and neural diseases are closely relatedand intertwined. Blood vessels depend on normal nerve function, andnerves depends on adequate blood flow. The first pathological change inthe microvasculature is vasoconstriction. As the disease progresses,neuronal dysfunction correlates closely with the development of vascularabnormalities, such as capillary basement membrane thickening andendothelial hyperplasia, which contribute to diminished oxygen tensionand hypoxia. Vasodilator agents (e.g., angiotensin-converting-enzymeinhibitors, α1-antagonists) can lead to substantial improvements inneuronal blood flow, with corresponding improvements in nerve conductionvelocities.

Clinical Manifestations

Neuropathy affects all peripheral nerves: pain fibers, motor neurons,autonomic nerves. It therefore necessarily can affect all organs andsystems since all are innervated. There are several distinct syndromesbased on the organ systems and members affected, but these are by nomeans exclusive. A patient can have sensorimotor and autonomicneuropathy or any other combination.

Despite advances in the understanding of the metabolic causes ofneuropathy, treatments aimed at interrupting these pathologicalprocesses have been limited by side effects and lack of efficacy. Thus,treatments are symptomatic and do not address the underlying problems.Agents for pain caused by sensorimotor neuropathy include tricyclicantidepressants (TCAs), serotonin reuptake inhibitors (SSRIs) andantiepileptic drugs (AEDs). None of these agents reverse thepathological processes leading to diabetic neuropathy and none alter therelentless course of the illness. Thus, it would be useful to have apharmaceutical composition that could better treat these conditionsand/or alleviate the symptoms.

Additional Retinopathies

Retinal Microvasculopathy (AIDS Retinopathy)

Retinal microvasculopathy is seen in 100% of AIDS patients. It ischaracterized by intraretinal hemorrhages, microaneurysms, Roth spots,cotton-wool spots (microinfarctions of the nerve fiber layer) andperivascular sheathing. The etiology of the retinopathy is unknownthough it has been thought to be due to circulating immune complexes,local release of cytotoxic substances, abnormal hemorheology, and HIVinfection of endothelial cells. AIDS retinopathy is now so common thatcotton wool spots in a patient without diabetes or hypertension but atrisk for HIV should prompt the physician to consider viral testing.There is no specific treatment for AIDS retinopathy but its continuedpresence may prompt a physician to reexamine the efficacy of the HIVtherapy and patient compliance.

Bone Marrow Transplantation (BMT) Retinopathy

Bone marrow transplantation retinopathy was first reported in 1983. Ittypically occurs within six months, but it can occur as late as 62months after BMT. Risk factors such as diabetes and hypertension mayfacilitate the development of BMT retinopathy by heightening theischemic microvasculopathy. There is no known age, gender or racepredilection for development of BMT retinopathy. Patients present withdecreased visual acuity and/or visual field deficit. Posterior segmentfindings are typically bilateral and symmetric. Clinical manifestationsinclude multiple cotton wool spots, telangiectasia, microaneurysms,macular edema, hard exudates and retinal hemorrhages. Fluoresceinangiography demonstrates capillary nonperfusion and dropout,intraretinal microvascular abnormalities, microaneurysms and macularedema. Although the precise etiology of BMT retinopathy has not beenelucidated, it appears to be affected by several factors: cyclosporinetoxicity, total body irradiation (TBI), and chemotherapeutic agents.Cyclosporine is a powerful immunomodulatory agent that suppressesgraft-versus-host immune response. It may lead to endothelial cellinjury and neurologic side effects, and as a result, it has beensuggested as the cause of BMT retinopathy. However, BMT retinopathy candevelop in the absence of cyclosporine use, and cyclosporine has notbeen shown to cause BMT retinopathy in autologous or syngeneic bonemarrow recipients. Cyclosporine does not, therefore, appear to be thesole cause of BMT retinopathy. Total body irradiation (TBI) has alsobeen implicated as the cause of BMT retinopathy. Radiation injures theretinal microvasculature and leads to ischemic vasculopathy. Variablessuch as the total dose of radiation and the time interval betweenradiation and bone marrow ablation appear to be important. However, BMTretinopathy can occur in patients who did not receive TBI, and BMTretinopathy is not observed in solid organ transplant recipients whoreceived similar doses of radiation. Thus, TBI is not the sole cause,but it is another contributing factor in development of BMT retinopathy.Chemotherapeutic agents have been suggested as a potential contributingfactor in BMT retinopathy. Medications such as cisplatin, carmustine,and cyclophosphamide can cause ocular side effects includingpapilledema, optic neuritis, visual field deficit and corticalblindness. It has been suggested that these chemotherapeutic drugs maypredispose patients to radiation-induced retinal damages and enhance thedeleterious effect of radiation. In general, patients with BMTretinopathy have a good prognosis. The retinopathy usually resolveswithin two to four months after stopping or lowering the dosage ofcyclosporine. In one report, 69 percent of patients experienced completeresolution of the retinal findings, and 46 percent of patients fullyrecovered their baseline visual acuity. Because of the favorableprognosis and relatively non-progressive nature of BMT retinopathy,aggressive intervention is usually not necessary.

Ischemic Conditions

Ischemia can be divided into 2 categories: the first involves theaccelerated atherosclerosis that occurs commonly in patients withdiabetes, i.e., in the femoral, popliteal, and posterior tibialarteries. These vessels, often only 1 or 2 cm in diameter, can developatherosclerotic plaque, which seriously decreases blood flow. Afterlarge vessels become completely occluded, stroke, myocardial infarction,ischemia, and nonhealing diabetic foot ulcers can occur. This form ofischemia is essentially a large-vessel disease.

Post Stroke Dementia

25% of people have dementia after a stroke with many others developingdementia over the following 5 to 10 years. In addition, many individualsexperience more subtle impairments of their higher brain functions (suchas planning skills and speed of processing information) and are at veryhigh risk of subsequently developing dementia. Very small strokes in thedeep parts of the brain in this process (called microvascular disease)seem to be essential in the process leading to an identified pattern ofbrain atrophy specific to post-stroke dementia.

Ocular Ischemic Syndrome

Patients suffering from ocular ischemic syndrome (OIS) are generallyelderly, ranging in age from the 50s to 80s. Males are affected twice ascommonly as females. The patient is only rarely asymptomatic. Decreasedvision occurs at presentation in 90 percent of cases, and 40 percent ofpatients have attendant eye pain. There may also be an attendant orantecedent history of transient ischemic attacks or amaurosis fugax.Patients also have significant known or unknown systemic disease at thetime of presentation. The most commonly encountered systemic diseasesare hypertension, diabetes, ischemic heart disease, stroke, andperipheral vascular disease. To a lesser extent, patients manifest OISas a result of giant cell arteritis (GCA).

Unilateral findings are present in 80 percent of cases. Common findingsmay include advanced unilateral cataract, anterior segment inflammation,asymptomatic anterior chamber reaction, macular edema, dilated butnon-tortuous retinal veins, mid-peripheral dot and blot hemorrhages,cotton wool spots, exudates, and neovascularization of the disc andretina. There may also be spontaneous arterial pulsation, elevatedintraocular pressure, and neovascularization of the iris and angle withneovascular glaucoma (NVG). While the patient may exhibit anteriorsegment neovascularization, ocular hypotony may occur due to lowarterial perfusion to the ciliary body. Occasionally, there is visibleretinal emboli (Hollenhorst plaques).

The findings in OIS are caused by internal carotid artery atheromatousulceration and stenosis at the bifurcation of the common carotid artery.Five percent of patients with internal artery stenosis develop OIS.However, OIS only occurs if the degree of stenosis exceeds 90 percent.Stenosis of the carotid artery reduces perfusion pressure to the eye,resulting in the above-mentioned ischemic phenomena. Once stenosisreaches 90 percent, the perfusion pressure in the central retinal artery(CRA) drops only to 50 percent. Often, the reduced arterial pressuremanifests as spontaneous pulsation of the CRA. The findings are variableand may include any or all of the above findings.

Patients with OIS have significant systemic disease that must beassessed. Cardiac death is the primary cause of mortality in patientswith OIS—the five-year mortality rate is 40 percent. For this reason,patients with OIS must be referred to a cardiologist for completeserology, EKG, ECG, and carotid evaluation.

Microvascular Diseases of the Kidney

The kidney is involved in a number of discreet clinicopathologicconditions that affect systemic and renal microvasculature. Certain ofthese conditions are characterized by primary injury to endothelialcells, such as:

-   -   hemolytic-uremic syndrome (HUS) and thrombotic thrombocytopenic        purpura (TTP) HUS and TTP are closely related diseases        characterized by microangiopathic hemolytic anemia and variable        organ impairment Traditionally, the diagnosis of HUS is made        when renal failure is a predominant feature of the syndrome, as        is common in children. In adults, neurologic impairment        frequently predominates and the syndrome is then referred to as        TTP. Thrombotic microangiopathy is the underlying pathologic        lesion in both syndromes, and the clinical and laboratory        findings in patients with either HUS or TTP overlap to a large        extent. This has prompted several investigators to regard the        two syndromes as a continuum of a single disease entity.        Pathogenesis: Experimental data strongly suggest that        endothelial cell injury is the primary event in the pathogenesis        of HUS/TTP. Endothelial damage triggers a cascade of events that        includes local intravascular coagulation, fibrin deposition, and        platelet activation and aggregation. The end result is the        histopathologic finding of thrombotic microangiopathy common to        the different forms of the HUS/TTP syndrome. If HUS/TTP is left        untreated, the mortality rate approaches 90%. Supportive        therapy—including dialysis, antihypertensive medications, blood        transfusions, and management of neurologic        complications—contributes to the improved survival of patients        with HUS/TTP. Adequate fluid balance and bowel rest are        important in treating typical HUS associated with diarrhea.    -   radiation nephritis—The long-term consequences of renal        irradiation in excess of 2500 rad can be divided into five        clinical syndromes:        -   (i) Acute radiation nephritis occurs in approximately 40% of            patients after a latency period of 6 to 13 months. It is            characterized clinically by abrupt onset of hypertension,            proteinuria, edema, and progressive renal failure in most            cases leading to end-stage kidneys.        -   (ii) Chronic radiation nephritis, conversely, has a latency            period that varies between 18 months and 14 years after the            initial insult. It is insidious in onset and is            characterized by hypertension, proteinuria, and gradual loss            of renal function.        -   (iii) The third syndrome manifests 5 to 19 years after            exposure to radiation as benign proteinuria with normal            renal function        -   (iv) A fourth group of patients exhibits only benign            hypertension 2 to 5 years later and may have variable            proteinuria. Late malignant hypertension arises 18 months to            11 years after irradiation in patients with either chronic            radiation nephritis or benign hypertension. Removal of the            affected kidney reversed the hypertension. Radiation-induced            damage to the renal arteries with subsequent renovascular            hypertension has been reported.        -   (v) A syndrome of renal insufficiency analogous to acute            radiation nephritis has been observed in bone marrow            transplantation (BMT) patients who were treated with            total-body irradiation (TBI).

It has been reported that irradiation causes endothelial dysfunction butspares vascular smooth muscle cells in the early postradiation phase.Radiation could directly damage DNA, leading to decreased regenerationof these cells and denudement of the basement membrane in the glomerularcapillaries and tubules. How this initial insult eventually leads toglomerulosclerosis, tubule atrophy, and interstitial fibrosis isunclear. It is postulated that degeneration of the endothelial celllayer may result in intravascular thrombosis in capillaries and smallerarterioles. This intrarenal angiopathy would then explain theprogressive renal fibrosis and the hypertension that characterizeradiation nephritis. A recent study of irradiated mouse kidneys showed adose-dependent increase in leukocytes in the renal cortex, suggesting arole for inflammatory processes in radiation-induced nephritis.

In other kidney diseases, the microvasculature of the kidney is involvedin autoimmune disorders, such as systemic sclerosis (scleroderma).Kidney involvement in systemic sclerosis manifests as a slowlyprogressing chronic renal disease or as scleroderma renal crisis (SRC),which is characterized by malignant hypertension and acute azotemia. Itis postulated that SRC is caused by a Raynaud-like phenomenon in thekidney. Severe vasospasm leads to cortical ischemia and enhancedproduction of renin and angiotensin II, which in turn perpetuate renalvasoconstriction. Hormonal changes (pregnancy), physical and emotionalstress, or cold temperature may trigger the Raynaud-like arterialvasospasm. The role of the renin-angiotensin system in perpetuatingrenal ischemia is underscored by the significant benefit of ACEinhibitors in treating SRC. In patients with SRC who progress to severerenal insufficiency despite antihypertensive treatment, dialysis becomesa necessity. Both peritoneal dialysis and hemodialysis have beenemployed. The End-Stage Renal Disease (ESRD) Network report on 311patients with systemic sclerosis-induced ESRD dialyzed between 1983 and1985 revealed a 33% survival rate at 3 years.

The renal microcirculation can also be affected in sickle cell disease,to which the kidney is particularly susceptible because of the lowoxygen tension attained in the deep vessels of the renal medulla as aresult of countercurrent transfer of oxygen along the vasa recta. Thesmaller renal arteries and arterioles can also be the site ofthromboembolic injury from cholesterol-containing material dislodgedfrom the walls of the large vessels.

Taken as a group, diseases that cause transient or permanent occlusionof renal microvasculature uniformly result in disruption of glomerularperfusion, and hence of the glomerular filtration rate, therebyconstituting a serious threat to systemic homeostasis.

Acute Renal Failure (ARF)

ARF can be caused by microvascular or macrovascular disease (major renalartery occlusion or severe abdominal aortic disease). The classicmicrovascular diseases often present with microangiopathic hemolysis andacute renal failure occurring because of glomerular capillary thrombosisor occlusion, often with accompanying thrombocytopenia. Typical examplesof these diseases include:

-   -   a) Thrombotic thrombocytopenic purpura—The classic pentad in        thrombotic thrombocytopenic purpura includes fever, neurologic        changes, renal failure, microangiopathic hemolytic anemia and        thrombocytopenia.    -   b) Hemolytic uremic syndrome—Hemolytic uremic syndrome is        similar to thrombotic thrombocytopenic purpura but does not        present with neurologic changes.    -   c) HELLP syndrome (hemolysis, elevated liver enzymes and low        platelets). HELLP syndrome is a type of hemolytic uremic        syndrome that occurs in pregnant women with the addition of        transaminase elevations.

Acute renal failure can present in all medical settings but ispredominantly acquired in hospitals. The condition develops in 5 percentof hospitalized patients, and approximately 0.5 percent of hospitalizedpatients require dialysis. Over the past 40 years, the survival rate foracute renal failure has not improved, primarily because affectedpatients are now older and have more comorbid conditions. Infectionaccounts for 75 percent of deaths in patients with acute renal failure,and cardio-respiratory complications are the second most common cause ofdeath. Depending on the severity of renal failure, the mortality ratecan range from 7 percent to as high as 80 percent. Acute renal failurecan be divided into three categories: Prerenal, intrinsic and postrenalARF. Intrinsic ARF is subdivided into four categories: tubular disease,glomerular disease, vascular disease (includes microvascular) andinterstitial disease.

Progressive Renal Disease

There is evidence that progressive renal disease is characterized by aprogressive loss of the microvasculature. The loss of themicrovasculature correlates directly with the development of glomerularand tubulointerstitial scarring. The mechanism is mediated in part by areduction in the endothelial proliferative response, and this impairmentin capillary repair is mediated by alteration in the local expression ofboth angiogenic (vascular endothelial growth factor) and antiangiogenic(thrombospondin 1) factors in the kidney. The alteration in balance ofangiogenic growth factors is mediated by both macrophage-associatedcytokines (interleukin-1β) and vasoactive mediators. Finally, there isintriguing evidence that stimulation of angiogenesis and/or capillaryrepair may stabilize renal function and slow progression and that thisbenefit occurs independently of effects on BP or proteinuria.

For further information see Brenner & Rector's The Kidney, 7th ed.,Copyright © 2004 Elsevier: Chapter 33—Microvascular diseases of thekidney and also Tiwari and Vikrant Journal of Indian Academy of ClinicalMedicine Vol. 5, No. 1 Review Article—Sepsis and the Kidney.

In conclusion, current modes of therapy for the prevention and/ortreatment of COPD, macular degeneration and microvascular diseases areunsatisfactory and there is a need therefore to develop novel compoundsfor this purpose. All the diseases and indications disclosed hereinabove, as well as other diseases and conditions described herein such asMI may also be treated by the novel compounds of this invention.

RTP801

Gene RTP801, was first reported by the assignee of the instantapplication. U.S. Pat. Nos. 6,455,674, 6,555,667, and 6740738, allassigned to the assignee of the instant application, disclose and claimper se the RTP801 polynucleotide and polypeptide, and antibodiesdirected toward the polypeptide. RTP801 represents a unique gene targetfor hypoxia-inducible factor-1 (HIF-1) that may regulate hypoxia-inducedpathogenesis independent of growth factors such as VEGF.

The inventor of the instant invention has made discoveries leading tothe novel concept of inhibiting gene RTP801 with the purpose ofimproving various respiratory disorders.

The following patent applications and publications give aspects ofbackground information.

WO 2001070979 relates to nucleic acid markers which are overexpressed inovarian cancer cells.

U.S. Pat. No. 6,673,549 discloses a combination comprising cDNAs thatare differentially expressed in response to steroid treatment.

US application 2003165864 relates to cDNAs that are differentiallyexpressed in cells treated with a DNA demethylating agent.

US application 2003108871 relates to a composition comprising severalcDNAs that are differentially expressed in treated human C3A liver cellcultures, allegedly useful for treating liver disorders.

US application 2002119463 discloses a new composition, useful fortreating and diagnosing prostate cancer, said composition comprisinghuman cDNAs that are differentially expressed in prostate cancer.

WO 2004018999 discloses a method for assessing, characterizing,monitoring, preventing and treating cervical cancer.

EP 1394274 relates to a method of testing for bronchial asthma orchronic obstructive pulmonary disease by comparing the expression levelof a marker gene in a biological sample from a subject with theexpression level of the gene in a sample from a healthy subject.

WO 2002101075 relates to an isolated nucleic acid molecule useful fordetecting, characterizing, preventing and treating human cervicalcancers.

WO 2003010205 relates to inhibiting angiogenesis for treating woundhealing, retinopathy, ischemia, inflammation, microvasculopathy, bonehealing and skin inflammation.

WO 2002046465 relates to identifying a gene involved in disease fortreating hypoxia-regulated conditions.

WO 2002031111 relates to allegedly novel polypeptides and their encodedproteins, and many uses therefore are provided.

WO 2001012659 relates to nucleic acids useful in recombinant DNAmethodologies.

WO 2001077289 discloses six hundred and twenty three polynucleotidesderived from a variety of human tissue sources.

WO 2003101283 relates to a combination which comprises many cDNAs andproteins allegedly differentially expressed in respiratory disorders.

JP 2003259877 relates to many hepatic fibrosis disease markers.

Tzipora Shoshani, et al. Identification of a Novel Hypoxia-InducibleFactor 1-Responsive Gene, RTP801, Involved in Apoptosis. MOLECULAR ANDCELLULAR BIOLOGY, April 2002, p. 2283-2293; this paper, co-authored bythe inventor of the present invention, details the discovery of theRTP801 gene (a then novel HIF-1-dependent gene

Anat Brafman, et al. Inhibition of Oxygen-Induced Retinopathy inRTP801-Deficient Mice. Invest Ophthalmol Vis Sci. 2004 October; 45 (10):3796-805; also co-authored by the inventor of the present invention,this paper demonstrates that in RTP801 knock out mice, hyperoxia doesnot cause degeneration of the retinal capillary network.

Leif W. Ellisen, et al. REDD1, a Developmentally RegulatedTranscriptional Target of p63 and p53, Links p63 to Regulation ofReactive Oxygen Species. Molecular Cell, Vol. 10, 995-1005, November,2002; this paper demonstrates that overexpression of RTP801 (referred totherein as REDD1) leads to increased production of reactive oxygenspecies.

Richard D R, Berra E, and Pouyssegur J. Non-hypoxic pathway mediates theinduction of hypoxia-inducible factor 1 alpha in vascular smooth musclecells. J. Biol. Chem. 2000, Sep. 1; 275(35): 26765-71 this paperdemonstrates that HIF-1-dependent transcription may be induced byexcessive production of reactive oxygen species.

Rangasami T, et al., Genetic ablation of Nrf2 enhances susceptibility tocigarette smoke-induced emphysema in mice. Submitted to Journal ofClinical Investigation. This work relates to mice with a compromisedantoxidant defence (due to a germline inactivation of RTP801, thereintermed Nrf2).

SUMMARY OF THE INVENTION

The present invention provides novel methods and compositions fortreating microvascular disorders, macular degeneration, respiratorydisorders, and spinal cord injury or disease.

In one embodiment, novel molecules which inhibit RTP801 and can be usedto treat various diseases and indications are provided.

In another embodiment, the present invention provides a method oftreating a patient suffering from a microvascular disorder, maculardegeneration or a respiratory disorder, comprising administering to thepatient a pharmaceutical composition comprising an RTP801 inhibitor.

Another embodiment of the present invention concerns a method fortreating a patient suffering from COPD, comprising administering to thepatient a pharmaceutical composition comprising a therapeuticallyeffective amount of an RTP801 inhibitor. In one embodiment the inhibitoris an siRNA molecule, an antisense molecule, an antibody (such as aneutralizing antibody), a dominant negative peptide or a ribozyme.

Another embodiment of the present invention concerns a method fortreating a patient suffering from macular degeneration, comprisingadministering to the patient a pharmaceutical composition comprising atherapeutically effective amount of an RTP801 inhibitor. In oneembodiment the inhibitor is an siRNA molecule, an antisense molecule, anantibody (such as a neutralizing antibody), a dominant negative peptideor a ribozyme.

Another embodiment of the present invention concerns a method fortreating a patient suffering from a microvascular disorder, comprisingadministering to the patient a pharmaceutical composition comprising atherapeutically effective amount of an RTP801 inhibitor. In oneembodiment the inhibitor is an siRNA molecule, an antisense molecule, anantibody (such as a neutralizing antibody), a dominant negative peptideor a ribozyme.

An additional embodiment of the present invention provides for the useof a therapeutically effective amount of an RTP801 inhibitor for thepreparation of a medicament for promoting recovery in a patientsuffering from a respiratory disorder. In one embodiment the respiratorydisorder is COPD and the inhibitor is preferably an siRNA.

An additional embodiment of the present invention provides for the useof a therapeutically effective dose of an RTP801 inhibitor for thepreparation of a medicament for promoting recovery in a patientsuffering from macular degeneration. In one embodiment the maculardegeneration is AMD and the inhibitor is preferably an siRNA.

An additional embodiment of the present invention provides for the useof a therapeutically effective amount of an RTP801 inhibitor for thepreparation of a medicament for promoting recovery in a patientsuffering from a microvascular disorder. In one embodiment themicrovascular disorder is diabetic retinopathy and the inhibitor ispreferably an siRNA.

DETAILED DESCRIPTION OF THE INVENTION

The present invention, in some of its embodiments, concerns inhibitionof the RTP801 gene or polypeptide for the treatment of eye diseases,respiratory disorders and microvascular disorders, inter alia. As willbe described herein, the preferred inhibitors to be used with thepresent invention are biological molecules.

Without being bound by theory, the inventors of the present inventionhave found that RTP801 is involved in various disease states includingmicrovascular disorders, eye diseases, respiratory disorders, and spinalcord injury and disease, and it would be beneficial to inhibit RTP801 inorder to treat any of said diseases or disorders. Methods, molecules andcompositions which inhibit RTP801 are discussed herein at length, andany of said molecules and/or compositions may be beneficially employedin the treatment of a patient suffering from any of said conditions.

The present invention provides methods and compositions for inhibitingexpression of the RTP801 gene in vivo. In general, the method includesadministering oligoribonucleotides, such as small interfering RNAs(i.e., siRNAs) that are targeted to a particular mRNA and hybridise toit, or nucleic acid material that can produce siRNAs in a cell, in anamount sufficient to down-regulate expression of a target gene by an RNAinterference mechanism. In particular, the subject method can be used toinhibit expression of the RTP801 gene for treatment of respiratorydisorders, microvascular disorders or eye disorders.

Thus, in one embodiment the present invention provides for a method oftreating a patient suffering from a microvascular disorder, aeye diseaseor a respiratory disorder, comprising administering to the patient apharmaceutical composition comprising an RTP801 inhibitor in atherapeutically effective amount so as to thereby treat the patient. Theinvention further provides a method of treating a patient suffering froma microvascular disorder, aeye disease or respiratory disorder,comprising administering to the patient a pharmaceutical compositioncomprising an RTP801 inhibitor, in a dosage and over a period of timesufficient to promote recovery. The eye disease may be maculardegeneration such as age-related macular degeneration (AMD), inter alia.The microvascular disorder may be diabetic retinopathy or acute renalfailure, inter alia. The respiratory disorder may be chronic obstructivepulmonary disease (COPD), emphysema, chronic bronchitis, asthma and lungcancer, inter alia. The RTP801 inhibitor may be selected from a largevariety of molecules, including but not limited to compounds such aspolynucleotides, AS fragments, RNA molecules which target the RTP801gene mRNA such as ribozymes or siRNAs (such as the siRNAs of Tables A-Cand in particular, siRNA Nos:14, 22, 23, 25, 27, 39, 41, 42, 49 and 50of Table A), or expression vectors comprising them; polypeptides such asdominant negatives, antibodies (such as an antibody which specificallybinds to an epitope present within a polypeptide which comprisesconsecutive amino acids, the sequence of which is set forth in FIG. 2(SEQ ID No:2)), or, in some cases, enzymes. Additionally, the RTP801inhibitor may be a chemical inhibitor such as a small molecule, e.g.,chemical molecules with a low molecular weight e.g. a molecular weightbelow 2000 daltons. Specific RTP801 inhibitors are given below.

The present invention further provides a method for treating a patientsuffering from macular degeneration, COPD or diabetic retinopathy,comprising administering to the patient a pharmaceutical compositioncomprising a therapeutically effective dose of an RTP801 inhibitorcomprising a polynucleotide which specifically hybridizes to mRNAtranscribed from the RTP801 gene and/or down regulates the expression ofthe RTP801 gene so as to thereby treat the patient. The polynucleotidemay be an siRNA comprising consecutive nucleotides having a sequenceidentical to any one of the sequences set forth in Tables A-C (SEQ IDNOs:3-344) and in particular, siRNA Nos: 14, 22, 23, 25, 27, 39, 41, 42,49 and 50 of Table A.

Further, an additional embodiment of the present invention concerns amethod for treating a patient suffering from a microvascular disorder, arespiratory disorder or an eye disease, comprising administering to thepatient a pharmaceutical composition comprising a therapeuticallyeffective dose of an RTP801 inhibitor comprising an siRNA molecule,optionally an siRNA molecule detailed in any one of Tables A-C, in adosage and over a period of time so as to thereby treat the patient.

An additional method for treating a patient suffering from amicrovascular disorder, a respiratory disorder or an eye disease isprovided, comprising administering to the patient a pharmaceuticalcomposition comprising a therapeutically effective dose of an RNAmolecule which targets the RTP801 gene mRNA in a dosage and over aperiod of time so as to thereby treat the patient. The RNA molecule maybe an siRNA molecule, such as an siRNA molecule detailed in Tables A-Cand in particular, siRNA Nos:14, 22, 23, 25, 27, 39, 41, 42, 49 and 50of Table A, or a ribozyme.

The present invention further provides a method for treating a patientsuffering from a respiratory disorder, a microvascular disorder or aneye disease or any of the conditions disclosed herein, comprisingadministering to the patient a pharmaceutical composition comprising atherapeutically effective dose of an siRNA molecule which targets theRTP801 gene mRNA, optionally an siRNA molecule detailed in Tables A-C,in a dosage and over a period of time so as to thereby treat thepatient. Further, the eye disease may be macular degeneration such asage-related macular degeneration (AMD); the microvascular disorder maybe diabetic retinopathy or acute renal failure; the respiratory disordermay be COPD and the aspects of COPD being treated may comprise, but arenot limited to, emphysema, chronic bronchitis, or both.

“Treating a disease” refers to administering a therapeutic substanceeffective to ameliorate symptoms associated with a disease, to lessenthe severity or cure the disease, or to prevent the disease fromoccurring.

A “therapeutically effective dose” refers to an amount of apharmaceutical compound or composition which is effective to achieve animprovement in a patient or his physiological systems including, but notlimited to, improved survival rate, more rapid recovery, or improvementor elimination of symptoms, and other indicators as are selected asappropriate determining measures by those skilled in the art.

The methods of treating the diseases disclosed herein and included inthe present invention may include administering an RTP801 inhibitor inconjunction with an additional RTP801 inhibitor, a substance whichimproves the pharmacological properties of the active ingredient asdetailed below, or an additional compound known to be effective in thetreatment of the disease to be treated, such as macular degeneration,COPD, ARF, DR, inter alia. By “in conjunction with” is meant prior to,simultaneously or subsequent to. Further detail on exemplary conjoinedtherapies is given below.

In another embodiment, the present invention provides for the use of atherapeutically effective dose of an RTP801 inhibitor for thepreparation of a medicament for promoting recovery in a patientsuffering from macular degeneration, COPD, ARF, DR, or any other eyedisease, microvascular or respiratory condition as detailed above, andthe use of a therapeutically effective dose of an RTP801 inhibitor forthe preparation of a medicament for treating said diseases andconditions. In this embodiment, the RTP801 inhibitor may comprise apolynucleotide which comprises consecutive nucleotides having a sequencewhich comprises an antisense sequence to the sequence set forth in FIG.1 (SEQ ID No: 1). Additionally, the RTP801 inhibitor may be anexpression vector comprising a polynucleotide having a sequence which isan antisense sequence to the sequence set forth in FIG. 1 (SEQ ID No:1).The RTP801 inhibitor according to said uses may also be an antibody,such as a neutralizing antibody which specifically binds to an epitopepresent within a polypeptide which comprises consecutive amino acids,the sequence of which is set forth in FIG. 2 (SEQ ID No:2).Additionally, the RTP801 inhibitor may be an RNA molecule which targetsthe RTP801 gene mRNA optionally an siRNA, optionally an siRNA comprisingconsecutive nucleotides having a sequence identical to any one of thesequences set forth in Tables A-C (SEQ ID NOs:3-344) and in particular,siRNA Nos: 14, 22, 23, 25, 27, 39, 41, 42, 49 and 50 of Table A, or aribozyme.

Thus, according to the information disclosed herein, the RTP801inhibitor to be used with any of the methods disclosed herein, in any ofthe uses disclosed herein and in any of the pharmaceutical compositionsdisclosed herein, may be selected from the group consisting of an siRNAmolecule, a vector comprising an siRNA molecule, a vector which canexpress an siRNA molecule and any molecule which is endogenouslyprocessed into an siRNA molecule. As detailed herein, said siRNAmolecule is preferably an siRNA comprising consecutive nucleotideshaving a sequence identical to any one of the sequences set forth inTables A-C (SEQ ID NOs:3-344) and in particular, siRNA Nos:14, 22, 23,25, 27, 39, 41, 42, 49 and 50 of Table A.

“Respiratory disorder” refers to conditions, diseases or syndromes ofthe respiratory system including but not limited to pulmonary disordersof all types including chronic obstructive pulmonary disease (COPD),emphysema, chronic bronchitis, asthma and lung cancer, inter alia.Emphysema and chronic bronchitis may occur as part of COPD orindependently.

“Microvascular disorder” refers to any condition that affectsmicroscopic capillaries and lymphatics, in particular vasospasticdiseases, vasculitic diseases and lymphatic occlusive diseases. Examplesof microvascular disorders include, inter alia: eye disorders such asAmaurosis Fugax (embolic or secondary to SLE), apla syndrome, Prot CSand ATIII deficiency, microvascular pathologies caused by IV drug use,dysproteinemia, temporal arteritis, anterior ischemic optic neuropathy,optic neuritis (primary or secondary to autoimmune diseases), glaucoma,von hippel lindau syndrome, corneal disease, corneal transplantrejection cataracts, Eales' disease, frosted branch angiitis, encirclingbuckling operation, uveitis including pars planitis, choroidal melanoma,choroidal hemangioma, optic nerve aplasia; retinal conditions such asretinal artery occlusion, retinal vein occlusion, retinopathy ofprematurity, HIV retinopathy, Purtscher retinopathy, retinopathy ofsystemic vasculitis and autoimmune diseases, diabetic retinopathy,hypertensive retinopathy, radiation retinopathy, branch retinal arteryor vein occlusion, idiopathic retinal vasculitis, aneurysms,neuroretinitis, retinal embolization, acute retinal necrosis, Birdshotretinochoroidopathy, long-standing retinal detachment; systemicconditions such as Diabetes mellitus, diabetic retinopathy (DR),diabetes-related microvascular pathologies (as detailed herein),hyperviscosity syndromes, aortic arch syndromes and ocular ischemicsyndromes, carotid-cavernous fistula, multiple sclerosis, systemic lupuserythematosus, arteriolitis with SS-A autoantibody, acute multifocalhemorrhagic vasculitis, vasculitis resulting from infection, vasculitisresulting from Behcet's disease, sarcoidosis, coagulopathies,neuropathies, nephropathies, microvascular diseases of the kidney, andischemic microvascular conditions, inter alia

Microvascular disorders may comprise a neovascular element. The term“neovascular disorder” refers to those conditions where the formation ofblood vessels (neovascularization) is harmful to the patient. Examplesof ocular neovascularization include: retinal diseases (diabeticretinopathy, diabetic Macular Edema, chronic glaucoma, retinaldetachment, and sickle cell retinopathy); rubeosis iritis; proliferativevitreo-retinopathy; inflammatory diseases; chronic uveitis; neoplasms(retinoblastoma, pseudoglioma and melanoma); Fuchs' heterochromiciridocyclitis; neovascular glaucoma; corneal neovascularization(inflammatory, transplantation and developmental hypoplasia of theiris); neovascularization following a combined vitrectomy andlensectomy; vascular diseases (retinal ischemia, choroidal vascularinsufficiency, choroidal thrombosis and carotid artery ischemia);neovascularization of the optic nerve; and neovascularization due topenetration of the eye or contusive ocular injury. All these neovascularconditions may be treated using the compounds and pharmaceuticalcompositions of the present invention.

“Eye disease” refers to refers to conditions, diseases or syndromes ofthe eye including but not limited to any conditions involving choroidalneovascularization (CNV), wet and dry AMD, ocular histoplasmosissyndrome, angiod streaks, ruptures in Bruch's membrane, myopicdegeneration, ocular tumors, retinal degenerative diseases and retinalvein occlusion (RVO). Some conditions disclosed herein, such as DR,which may be treated according to the methods of the present inventionhave been regarded as either a microvascular disorder and an eyedisease, or both, under the definitions presented herein.

“RTP801 gene” refers to the RTP801 coding sequence open reading frame,as shown in FIG. 1 (SEQ ID NO:1), or any homologous sequence thereofpreferably having at least 70% identity, more preferable 80% identity,even more preferably 90% or 95% identity. This encompasses any sequencesderived from SEQ ID NO:1 which have undergone mutations, alterations ormodifications as described herein. Thus, in a preferred embodimentRTP801 is encoded by a nucleic acid sequence according to SEQ. ID.NO. 1. It is also within the present invention that the nucleic acidsaccording to the present invention are only complementary and identical,respectively, to a part of the nucleic acid coding for RTP801 as,preferably, the first stretch and first strand is typically shorter thanthe nucleic acid according to the present invention. It is also to beacknowledged that based on the amino acid sequence of RTP801 any nucleicacid sequence coding for such amino acid sequence can be perceived bythe one skilled in the art based on the genetic code. However, due tothe assumed mode of action of the nucleic acids according to the presentinvention, it is most preferred that the nucleic acid coding for RTP801,preferably the mRNA thereof, is the one present in the organism, tissueand/or cell, respectively, where the expression of RTP801 is to bereduced.

“RTP801 polypeptide” refers to the polypeptide of the RTP801 gene, andis understood to include, for the purposes of the instant invention, theterms “RTP779”, “REDD1”, “Ddit4”, “FLJ20500”, “Dig2”, and “PRF1”,derived from any organism, optionally man, splice variants and fragmentsthereof retaining biological activity, and homologs thereof, preferablyhaving at least 70%, more preferably at least 80%, even more preferablyat least 90% or 95% homology thereto. In addition, this term isunderstood to encompass polypeptides resulting from minor alterations inthe RTP801 coding sequence, such as, inter alia, point mutations,substitutions, deletions and insertions which may cause a difference ina few amino acids between the resultant polypeptide and the naturallyoccurring RTP801. Polypeptides encoded by nucleic acid sequences whichbind to the RTP801 coding sequence or genomic sequence under conditionsof highly stringent hybridization, which are well-known in the art (forexample Ausubel et al., Current Protocols in Molecular Biology, JohnWiley and Sons, Baltimore, Md. (1988), updated in 1995 and 1998), arealso encompassed by this term. Chemically modified RTP801 or chemicallymodified fragments of RTP801 are also included in the term, so long asthe biological activity is retained. RTP801 preferably has or comprisesan amino acid sequence according to SEQ. ID. NO. 2. It is acknowledgedthat there might be differences in the amino acid sequence among varioustissues of an organism and among different organisms of one species oramong different species to which the nucleic acid according to thepresent invention can be applied in various embodiments of the presentinvention. However, based on the technical teaching provided herein, therespective sequence can be taken into consideration accordingly whendesigning any of the nucleic acids according to the present invention.Particular fragments of RTP801 include amino acids 1-50, 51-100,101-150, 151-200 and 201-232 of the sequence shown in FIG. 2. Furtherparticular fragments of RTP801 include amino acids 25-74, 75-124,125-174, 175-224 and 225-232 of the sequence shown in FIG. 2.

RTP801 as used herein is a protein described, among others, in WO99/09046. RTP801 which is also referred to as RTP801, has been describedas a transcriptional target of HIF-1α by Shoshani T et al. (Shoshani etal., 2002, Mol Cell Biol, 22, 2283-93). Furthermore the study by Ellisenet al. (Ellisen et al., Mol Cell, 10, 995-1005) has identified RTP801 asa p53-dependent DNA damage response gene and as a p63-dependent geneinvolved in epithelial differentiation. Also, RTP801 mirrors thetissue-specific pattern of the p53 family member p63, is effectivesimilar to or in addition to TP 63, is an inhibitor to in vitrodifferentiation, and is involved in the regulation of reactive oxygenspecies. Apart from that, RTP801 is responsive to hypoxia-responsivetranscription factor hypoxia-inducible factor 1 (HIF-1) and is typicallyup-regulated during hypoxia both in vitro and in vivo in an animal modelof ischemic stroke. RTP801 appears to function in the regulation ofreactive oxygen species (ROS) and ROS levels and reduced sensitivity tooxidative stress are both increased following ectopic expression RTP801(Ellisen et al. 2002, supra; Soshani et al. 2002, supra). Preferably,RTP801 is a biologically active RTP801 protein which preferably exhibitsat least one of those characteristics, preferable two or more and mostpreferably each and any of these characteristics.

A related gene to RTP801 is RT801L, also referred to as “REDD2”, wasdiscovered by the inventors of the present invention. RTP801L ishomologous to RTP801, and reacts in a similar manner to oxidativestress; thus, RTP801L probably possesses some similar functions withRTP801.

Without being bound by theory, RTP801 being a stress-inducible protein(responding to hypoxia, oxidative stress, termal stress, ER stress) is afactor acting in fine-tuning of cell response to energy disbalance. Assuch, it is a target suitable for treatment of any disease where cellsshould be rescued from apoptosis due to stressful conditions (e.g.diseases accompanied by death of normal cells) or where cells, which areadapted to stressful conditions due to changes in RTP801 expression(e.g. cancer cells), should be killed. In the latter case, RTP801 may beviewed as a survival factor for cancer cells and its inhibitors maytreat cancer as a monotherapy or as sensitising drugs in compbinationwith chemotherapy or radiotherapy.

The term “polynucleotide” refers to any molecule composed of DNAnucleotides, RNA nucleotides or a combination of both types, i.e. thatcomprises two or more of the bases guanidine, cytosine, thymidine,adenine, uracil or inosine, inter alia. A polynucleotide may includenatural nucleotides, chemically modified nucleotides and syntheticnucleotides, or chemical analogs thereof. The term includes“oligonucleotides” and encompasses “nucleic acids”.

The term “amino acid” refers to a molecule which consists of any one ofthe 20 naturally occurring amino acids, amino acids which have beenchemically modified (see below), or synthetic amino acids.

The term “polypeptide” refers to a molecule composed of two or moreamino acids residues.

The term includes peptides, polypeptides, proteins and peptidomimetics.

A “peptidomimetic” is a compound containing non-peptidic structuralelements that is capable of mimicking the biological action(s) of anatural parent peptide. Some of the classical peptide characteristicssuch as enzymatically scissille peptidic bonds are normally not presentin a peptidomimetic.

By the term “dominant negative peptide” is meant a polypeptide encodedby a cDNA fragment that encodes for a part of a protein (see HerskowitzI.: Functional inactivation of genes by dominant negative mutations.Nature. 1987 Sep. 17-23; 329(6136):219-22. Review; Roninson I B et al.,Genetic suppressor elements: new tools for molecular oncology—thirteenthCornelius P. Rhoads Memorial Award Lecture. Cancer Res. 1995 Sep. 15;55(18):4023). This peptide can have a different function from theprotein from which it was derived. It can interact with the full proteinand inhibit its activity or it can interact with other proteins andinhibit their activity in response to the full-length (parent) protein.Dominant negative means that the peptide is able to overcome the naturalparent protein and inhibit its activity to give the cell a differentcharacteristic, such as resistance or sensitization to death or anycellular phenotype of interest. For therapeutic intervention the peptideitself may be delivered as the active ingredient of a pharmaceuticalcomposition, or the cDNA can be delivered to the cell utilizing knownmethods.

Preparation of Peptides and Polypeptides

Polypeptides may be produced via several methods, for example:

1) Synthetically:

Synthetic polypeptides can be made using a commercially availablemachine, using the known sequence of RTP801 or a portion thereof.

2) Recombinant Methods:

A preferred method of making the RTP801 polypeptides of fragmentsthereof is to clone a polynucleotide comprising the cDNA of the RTP801gene into an expression vector and culture the cell harboring the vectorso as to express the encoded polypeptide, and then purify the resultingpolypeptide, all performed using methods known in the art as describedin, for example, Marshak et al., “Strategies for Protein Purificationand Characterization. A laboratory course manual.” CSHL Press (1996).(in addition, see Bibl Haematol. 1965; 23:1165-74 Appl Microbiol. 1967July; 15(4):851-6; Can J Biochem. 1968 May; 46(5):441-4; Biochemistry.1968 July; 7(7):2574-80; Arch Biochem Biophys. 1968 Sep. 10;126(3):746-72; Biochem Biophys Res Commun. 1970 Feb. 20;38(4):825-30).).

The expression vector can include a promoter for controllingtranscription of the heterologous material and can be either aconstitutive or inducible promoter to allow selective transcription.Enhancers that can be required to obtain necessary transcription levelscan optionally be included. The expression vehicle can also include aselection gene.

Vectors can be introduced into cells or tissues by any one of a varietyof methods known within the art. Such methods can be found generallydescribed in Sambrook et al., Molecular Cloning: A Laboratory Manual,Cold Springs Harbor Laboratory, New York (1989, 1992), in Ausubel etal., Current Protocols in Molecular Biology, John Wiley and Sons,Baltimore, Md. (1989), Vega et al., Gene Targeting, CRC Press, AnnArbor, Mich. (1995), Vectors: A Survey of Molecular Cloning Vectors andTheir Uses, Butterworths, Boston Mass. (1988) and Gilboa et al. (1986).

3) Purification from Natural Sources:

RTP801 polypeptide, or naturally occurring fragments thereof, can bepurified from natural sources (such as tissues) using many methods knownto one of ordinary skill in the art, such as for example:immuno-precipitation with anti-RTP801 antibody, or matrix-bound affinitychromatography with any molecule known to bind RTP801.

Protein purification is practiced as is known in the art as describedin, for example, Marshak et al., “Strategies for Protein Purificationand Characterization. A laboratory course manual.” CSHL Press (1996).

By “biological effect of RTP801” or “RTP801 biological activity” ismeant the effect of RTP801 in respiratory disorders, which may be director indirect, and includes, without being bound by theory, the effect ofRTP801 on apoptosis of alveolar cells induced by hypoxic or hyperoxicconditions. The indirect effect includes, but is not limited to, RTP801binding to or having an effect on one of several molecules, which areinvolved in a signal transduction cascade resulting in apoptosis.

“Apoptosis” refers to a physiological type of cell death which resultsfrom activation of some cellular mechanisms, i.e. death that iscontrolled by the machinery of the cell. Apoptosis may, for example, bethe result of activation of the cell machinery by an external trigger,e.g. a cytokine or anti-FAS antibody, which leads to cell death or by aninternal signal. The term “programmed cell death” may also be usedinterchangeably with “apoptosis”.

“Apoptosis-related disease” refers to a disease whose etiology isrelated either wholly or partially to the process of apoptosis. Thedisease may be caused either by a malfunction of the apoptotic process(such as in cancer or an autoimmune disease) or by overactivity of theapoptotic process (such as in certain neurodegenerative diseases). Manydiseases in which RTP801 is involved are apoptosis-related diseases. Forexample, apoptosis is a significant mechanism in dry AMD, whereby slowatrophy of photoreceptor and pigment epithelium cells, primarily in thecentral (macular) region of retina takes place. Neuroretinal apoptosisis also a significant mechanism in diabetic retinopathy.

An “inhibitor” is a compound which is capable of inhibiting the activityof a gene or the product of such gene to an extent sufficient to achievea desired biological or physiological effect. An “RTP801 inhibitor” is acompound which is capable of inhibiting the activity of the RTP801 geneor RTP801 gene product, particularly the human RTP801 gene or geneproduct. Such inhibitors include substances that affect thetranscription or translation of the gene as well as substances thataffect the activity of the gene product. An RTP801 inhibitor may also bean inhibitor of the RTP801 promoter. Examples of such inhibitors mayinclude, inter alia: polynucleotides such as AS fragments, siRNA, orvectors comprising them; polypeptides such as dominant negatives,antibodies, and enzymes; catalytic RNAs such as ribozymes; and chemicalmolecules with a low molecular weight e.g. a molecular weight below 2000daltons. Specific RTP801 inhibitors are given below.

“Expression vector” refers to a vector that has the ability toincorporate and express heterologous DNA fragments in a foreign cell.Many prokaryotic and eukaryotic expression vectors are known and/orcommercially available. Selection of appropriate expression vectors iswithin the knowledge of those having skill in the art.

The term “antibody” refers to IgG, IgM, IgD, IgA, and IgE antibody,inter alia. The definition includes polyclonal antibodies or monoclonalantibodies. This term refers to whole antibodies or fragments ofantibodies comprising an antigen-binding domain, e.g. antibodies withoutthe Fc portion, single chain antibodies, miniantibodies, fragmentsconsisting of essentially only the variable, antigen-binding domain ofthe antibody, etc. The term “antibody” may also refer to antibodiesagainst polynucleotide sequences obtained by cDNA vaccination. The termalso encompasses antibody fragments which retain the ability toselectively bind with their antigen or receptor and are exemplified asfollows, inter alia:

-   -   (1) Fab, the fragment which contains a monovalent        antigen-binding fragment of an antibody molecule which can be        produced by digestion of whole antibody with the enzyme papain        to yield a light chain and a portion of the heavy chain;    -   (2) (Fab′)₂, the fragment of the antibody that can be obtained        by treating whole antibody with the enzyme pepsin without        subsequent reduction; F(ab′₂) is a dimer of two Fab fragments        held together by two disulfide bonds;    -   (3) Fv, defined as a genetically engineered fragment containing        the variable region of the light chain and the variable region        of the heavy chain expressed as two chains; and    -   (4) Single chain antibody (SCA), defined as a genetically        engineered molecule containing the variable region of the light        chain and the variable region of the heavy chain linked by a        suitable polypeptide linker as a genetically fused single chain        molecule.

By the term “epitope” as used in this invention is meant an antigenicdeterminant on an antigen to which the antibody binds. Epitopicdeterminants usually consist of chemically active surface groupings ofmolecules such as amino acids or sugar side chains and usually havespecific three-dimensional structural characteristics, as well asspecific charge characteristics.

Preparation of Anti-RTP801 Antibodies

Antibodies which bind to RTP801 or a fragment derived therefrom may beprepared using an intact polypeptide or fragments containing smallerpolypeptides as the immunizing antigen. For example, it may be desirableto produce antibodies that specifically bind to the N- or C-terminal orany other suitable domains of the RTP801. The polypeptide used toimmunize an animal can be derived from translated cDNA or chemicalsynthesis and can be conjugated to a carrier protein, if desired. Suchcommonly used carriers which are chemically coupled to the polypeptideinclude keyhole limpet hemocyanin (KLH), thyroglobulin, bovine serumalbumin (BSA) and tetanus toxoid. The coupled polypeptide is then usedto immunize the animal.

If desired, polyclonal or monoclonal antibodies can be further purified,for example by binding to and elution from a matrix to which thepolypeptide or a peptide to which the antibodies were raised is bound.Those skilled in the art know various techniques common in immunologyfor purification and/or concentration of polyclonal as well asmonoclonal antibodies (Coligan et al, Unit 9, Current Protocols inImmunology, Wiley Interscience, 1994).

Methods for making antibodies of all types, including fragments, areknown in the art (See for example, Harlow and Lane, Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory, New York (1988)).Methods of immunization, including all necessary steps of preparing theimmunogen in a suitable adjuvant, determining antibody binding,isolation of antibodies, methods for obtaining monoclonal antibodies,and humanization of monoclonal antibodies are all known to the skilledartisan.

The antibodies may be humanized antibodies or human antibodies.Antibodies can be humanized using a variety of techniques known in theart including CDR-grafting (EP239,400: PCT publication WO.91/09967; U.S.Pat. Nos. 5,225,539; 5,530,101; and 5,585,089, veneering or resurfacing(EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498(1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994);Roguska et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat.No. 5,565,332).

The monoclonal antibodies as defined include antibodies derived from onespecies (such as murine, rabbit, goat, rat, human, etc.) as well asantibodies derived from two (or more) species, such as chimeric andhumanized antibodies.

Completely human antibodies are particularly desirable for therapeutictreatment of human patients. Human antibodies can be made by a varietyof methods known in the art including phage display methods usingantibody libraries derived from human immunoglobulin sequences. See alsoU.S. Pat. Nos. 4,444,887 and 4,716,111; and PCT publications WO98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO96/33735, and WO 91/10741, each of which is incorporated herein byreference in its entirety.

Additional information regarding all types of antibodies, includinghumanized antibodies, human antibodies and antibody fragments can befound in WO 01/05998, which is incorporated herein by reference in itsentirety.

Neutralizing antibodies can be prepared by the methods discussed above,possibly with an additional step of screening for neutralizing activityby, for example, a survival assay.

The terms “chemical compound”, “small molecule”, “chemical molecule”“small chemical molecule” and “small chemical compound” are usedinterchangeably herein and are understood to refer to chemical moietiesof any particular type which may be synthetically produced or obtainedfrom natural sources and usually have a molecular weight of less than2000 daltons, less than 1000 daltons or even less than 600 daltons.

The present invention also relates to functional nucleic acidscomprising a double-stranded structure, their use for the manufacture ofa medicament, a pharmaceutical composition comprising such functionalnucleic acids and a method for the treatment of a patient.

Hypoxia has been recognised as a key element in the pathomechanism ofquite a number of diseases such as stroke, emphysema and infarct whichare associated with sub-optimum oxygen availability and tissue damagingresponses to the hypoxia conditions. In fast-growing tissues, includingtumor, a sub-optimum oxygen availability is compensated by undesiredneo-angiogenesis. Therefore, at least in case of cancer diseases, thegrowth of vasculature is undesired.

In view of this, the inhibition of angiogenesis and vascular growth,respectively, is subject to intense research. Already today somecompounds are available which inhibit undesired angiogenesis andvascular growth. Some of the more prominent compounds are thoseinhibiting VEGF and the VEGF receptor. In both cases, the effect of VEGFis avoided by either blocking VEGF as such, for example by using anantibody directed against VEGF such as pursued by Genentech's AVASTIN(monoclonal AB specific for VEGF) (Ferrara N.; Endocr Rev. 2004 August;25(4):581-611), or by blocking the corresponding receptor, i.e. the VEGFreceptor (Traxler P; Cancer Res. 2004 Jul. 15; 64(14):4931-41; orStadler W M et al., Clin Cancer Res. 2004 May 15; 10(10):3365-70).

As, however, angiogenesis and the growth of vasculature is a very basicand vital process in any animal and human being, the effect of this kindof compound has to be focused at the particular site where angiogenesisand vascular growth is actually undesired which renders appropriatetargeting or delivery a critical issue in connection with this kind oftherapeutic approach.

It is thus an objective of the present invention to provide furthermeans for the treatment of diseases involving undesired growth ofvasculature and angiogenesis, respectively.

By “small interfering RNA” (siRNA) is meant an RNA molecule whichdecreases or silences (prevents) the expression of a gene/mRNA of itsendogenous cellular counterpart. The term is understood to encompass“RNA interference” (RNAi). RNA interference (RNAi) refers to the processof sequence-specific post transcriptional gene silencing in mammalsmediated by small interfering RNAs (siRNAs) (Fire et al, 1998, Nature391, 806). The corresponding process in plants is commonly referred toas specific post transcriptional gene silencing or RNA silencing and isalso referred to as quelling in fungi. The RNA interference response mayfeature an endonuclease complex containing an siRNA, commonly referredto as an RNA-induced silencing complex (RISC), which mediates cleavageof single-stranded RNA having sequence complementary to the antisensestrand of the siRNA duplex. Cleavage of the target RNA may take place inthe middle of the region complementary to the antisense strand of thesiRNA duplex (Elbashir et al 2001, Genes Dev., 15, 188). For recentinformation on these terms and proposed mechanisms, see Bernstein E.,Denli A M., Hannon G J: The rest is silence. RNA. 2001 November;7(11):1509-21; and Nishikura K.: A short primer on RNAi: RNA-directedRNA polymerase acts as a key catalyst. Cell. 2001 Nov. 16; 107(4):415-8.Examples of siRNA molecules which may be used in the present inventionare given in Tables A-C.

During recent years, RNAi has emerged as one of the most efficientmethods for inactivation of genes (Nature Reviews, 2002, v.3, p. 737-47;Nature, 2002, v.418, p. 244-51). As a method, it is based on the abilityof dsRNA species to enter a specific protein complex, where it is thentargeted to the complementary cellular RNA and specifically degrades it.In more detail, dsRNAs are digested into short (17-29 bp) inhibitoryRNAs (siRNAs) by type III RNAses (DICER, Drosha, etc) (Nature, 2001,v.409, p. 363-6; Nature, 2003, 425, p. 415-9). These fragments andcomplementary mRNA are recognized by the specific RISC protein complex.The whole process is culminated by endonuclease cleavage of target mRNA(Nature Reviews, 2002, v.3, p. 737-47; Curr Opin Mol Ther. 2003 June;5(3):217-24).

For disclosure on how to design and prepare siRNA to known genes see forexample Chalk A M, Wahlestedt C, Sonnhammer E L. Improved and automatedprediction of effective siRNA Biochem. Biophys. Res. Commun. 2004 Jun.18; 319(1):264-74; Sioud M, Leirdal M., Potential design rules andenzymatic synthesis of siRNAs, Methods Mol. Biol. 2004; 252:457-69;Levenkova N, Gu Q, Rux J J.: Gene specific siRNA selectorBioinformatics. 2004 Feb. 12; 20(3):430-2. and Ui-Tei K, Naito Y,Takahashi F, Haraguchi T, Ohki-Hamazaki H, Juni A, Ueda R, Saigo K.,Guidelines for the selection of highly effective siRNA sequences formammalian and chick RNA interference Nucleic Acids Res. 2004 Feb. 9;32(3):936-48. See also Liu Y, Braasch D A, Nulf C J, Corey D R.Efficient and isoform-selective inhibition of cellular gene expressionby peptide nucleic acids Biochemistry, 2004 Feb. 24; 43(7):1921-7. Seealso PCT publications WO 2004/015107 (Atugen) and WO 02/44321 (Tuschl etal), and also Chiu Y L, Rana T M. siRNA function in RNAi: a chemicalmodification analysis, RNA 2003 September; 9(9):1034-48 and U.S. Pat.Nos. 5,898,031 and 6,107,094 (Crooke) for production of modified/morestable siRNAs.

DNA-based vectors capable of generating siRNA within cells have beendeveloped. The method generally involves transcription of short hairpinRNAs that are efficiently processed to form siRNAs within cells.Paddison et al. PNAS 2002, 99:1443-1448; Paddison et al. Genes & Dev2002, 16:948-958; Sui et al. PNAS 2002, 8:5515-5520; and Brummelkamp etal. Science 2002, 296:550-553. These reports describe methods togenerate siRNAs capable of specifically targeting numerous endogenouslyand exogenously expressed genes.

For delivery of siRNAs, see, for example, Shen et al (FEBS letters 539:111-114 (2003)), Xia et al., Nature Biotechnology 20: 1006-1010 (2002),Reich et al., Molecular Vision 9: 210-216 (2003), Sorensen et al. (J.Mol. Biol. 327: 761-766 (2003), Lewis et al., Nature Genetics 32:107-108 (2002) and Simeoni et al., Nucleic Acids Research 31, 11:2717-2724 (2003). siRNA has recently been successfully used forinhibition in primates; for further details see Tolentino et al., Retina24(1) February 2004 pp 132-138.

siRNAs of the Present Invention

General Specifications of siRNAs of the Present Invention

Generally, the siRNAs used in the present invention comprise aribonucleic acid comprising a double stranded structure, whereby thedouble-stranded structure comprises a first strand and a second strand,whereby the first strand comprises a first stretch of contiguousnucleotides and whereby said first stretch is at least partiallycomplementary to a target nucleic acid, and the second strand comprisesa second stretch of contiguous nucleotides and whereby said secondstretch is at least partially identical to a target nucleic acid,whereby said first strand and/or said second strand comprises aplurality of groups of modified nucleotides having a modification at the2′-position whereby within the strand each group of modified nucleotidesis flanked on one or both sides by a flanking group of nucleotideswhereby the flanking nucleotides forming the flanking group ofnucleotides is either an unmodified nucleotide or a nucleotide having amodification different from the modification of the modifiednucleotides. Further, said first strand and/or said second strand maycomprise said plurality of modified nucleotides and may comprises saidplurality of groups of modified nucleotides.

The group of modified nucleotides and/or the group of flankingnucleotides may comprise a number of nucleotides whereby the number isselected from the group comprising one nucleotide to 10 nucleotides. Inconnection with any ranges specified herein it is to be understood thateach range discloses any individual integer between the respectivefigures used to define the range including said two figures definingsaid range. In the present case the group thus comprises one nucleotide,two nucleotides, three nucleotides, four nucleotides, five nucleotides,six nucleotides, seven nucleotides, eight nucleotides, nine nucleotidesand ten nucleotides.

The pattern of modified nucleotides of said first strand may be the sameas the pattern of modified nucleotides of said second strand, and mayalign with the pattern of said second strand. Additionally, the patternof said first strand may be shifted by one or more nucleotides relativeto the pattern of the second strand.

The modifications discussed above may be selected from the groupcomprising amino, fluoro, methoxy, alkoxy and alkyl.

The double stranded structure of the siRNA may be blunt ended, on one orboth sides. More specifically, the double stranded structure may beblunt ended on the double stranded structure's side which is defined bythe S′-end of the first strand and the 3′-end of the second strand, orthe double stranded structure may be blunt ended on the double strandedstructure's side which is defined by at the 3′-end of the first strandand the 5′-end of the second strand.

Additionally, at least one of the two strands may have an overhang of atleast one nucleotide at the 5′-end; the overhang may consist of at leastone deoxyribonucleotide. At least one of the strands may also optionallyhave an overhang of at least one nucleotide at the 3′-end.

The length of the double-stranded structure of the siRNA is typicallyfrom about 17 to 21 and more preferably 18 or 19 bases. Further, thelength of said first strand and/or the length of said second strand mayindependently from each other be selected from the group comprising theranges of from about 15 to about 23 bases, 17 to 21 bases and 18 or 19bases.

Additionally, the complementarily between said first strand and thetarget nucleic acid may be perfect, or the duplex formed between thefirst strand and the target nucleic acid may comprise at least 15nucleotides wherein there is one mismatch or two mismatches between saidfirst strand and the target nucleic acid forming said double-strandedstructure.

In some cases both the first strand and the second strand each compriseat least one group of modified nucleotides and at least one flankinggroup of nucleotides, whereby each group of modified nucleotidescomprises at least one nucleotide and whereby each flanking group ofnucleotides comprising at least one nucleotide with each group ofmodified nucleotides of the first strand being aligned with a flankinggroup of nucleotides on the second strand, whereby the most terminal S′nucleotide of the first strand is a nucleotide of the group of modifiednucleotides, and the most terminal 3′ nucleotide of the second strand isa nucleotide of the flanking group of nucleotides. Each group ofmodified nucleotides may consist of a single nucleotide and/or eachflanking group of nucleotides may consist of a single nucleotide.

Additionally, it is possible that on the first strand the nucleotideforming the flanking group of nucleotides is an unmodified nucleotidewhich is arranged in a 3′ direction relative to the nucleotide formingthe group of modified nucleotides, and on the second strand thenucleotide forming the group of modified nucleotides is a modifiednucleotide which is arranged in 5′ direction relative to the nucleotideforming the flanking group of nucleotides.

Further the first strand of the siRNA may comprise eight to twelve,preferably nine to eleven, groups of modified nucleotides, and thesecond strand may comprise seven to eleven, preferably eight to ten,groups of modified nucleotides.

The first strand and the second strand may be linked by a loopstructure, which may be comprised of a non-nucleic acid polymer such as,inter alia, polyethylene glycol. Alternatively, the loop structure maybe comprised of a nucleic acid.

Further, the 5′-terminus of the first strand of the siRNA may be linkedto the 3′-terminus of the second strand, or the 3′-end of the firststrand may be linked to the 5′-terminus of the second strand, saidlinkage being via a nucleic acid linker typically having a lengthbetween 10-2000 nucleobases.

Particular Specifications of siRNAs of the Present Invention

The invention provides a compound having the structure (structure A):5′(N)_(x)-Z3′ (antisense strand)3′Z′-(N′)_(y)5′ (sense strand)

-   -   wherein each N and N′ is a ribonucleotide which may be modified        or unmodified in its sugar residue and (N)_(x) and (N′)_(y) is        oligomer in which each consecutive N or N′ is joined to the next        N or N′ by a covalent bond;    -   wherein each of x and y is an integer between 19 and 40;    -   wherein each of Z and Z′ may be present or absent, but if        present is dTdT and is covalently attached at the 3′ terminus of        the strand in which it is present;    -   and wherein the sequence of (N)_(x) comprises an antisense        sequence to cDNA of the RTP801 gene

In particular, the invention provides the above compound wherein thesequence of (N)_(x) comprises one or more of the antisense sequencespresent in Tables A, B and C.

In particular, the invention provides the above compound wherein thecovalent bond is a phosphodiester bond, wherein x=y, preferably whereinx=y=19, wherein Z and Z′ are both absent, wherein at least oneribonucleotide is modified in its sugar residue at the 2′ position,wherein the moiety at the 2′ position is methoxy(2′-O-methyl) whereinalternating ribonucleotides are modified in both the antisense and thesense strands and wherein the ribonucleotides at the 5′ and 3′ terminiof the antisense strand are modified in their sugar residues, and theribonucleotides at the 5′ and 3′ termini of the sense strand areunmodified in their sugar residues.

In particular, the siRNA used in the present invention is anoligoribonucleotide wherein one strand comprises consecutive nucleotideshaving, from 5′ to 3′, the sequence set forth in SEQ ID NOS: 3-52 or inSEQ ID NOS: 103-174 or in SEQ ID NOS: 247-295 (which are sense strands)wherein a plurality of the bases may be modified, preferably by a2-O-methyl modification, or a homolog thereof wherein in up to 2 of thenucleotides in each terminal region a base is altered.

Further, the present invention provides for a method of treating apatient suffering from a respiratory disorder, an eye disease, amicrovascular disorder, or a spinal cord injury or disease comprisingadministering to the patient a pharmaceutical composition comprising acompound of the above structure (A) (having any of the specificsmentioned above) in a therapeutically effective amount so as to therebytreat the patient. Additionally, the invention provides for the use of atherapeutically effective amount of the above structure (A) (having anyof the specifics mentioned above) for the preparation of a medicamentfor promoting recovery in a patient suffering from a respiratorydisorder, an eye disease, a microvascular disorder or spinal cord injuryor disease.

An additional aspect of the present invention provides for apharmaceutical composition comprising a compound of the above structure(A) for the treatment of any of the diseases and conditions mentionedherein.

Further, this aspect provides for a pharmaceutical compositioncomprising two or more compounds of the above structure (A) for thetreatment of any of the diseases and conditions mentioned herein,whereby said two compounds may be physically mixed together in thepharmaceutical composition in amounts which generate equal or otherwisebeneficial activity, or may be covalently or non-covalently bound, orjoined together by a nucleic acid linker of a length ranging from 2-100,preferably 2-50 or 2-30 nucleotides. Such siRNA molecules are thereforecomprised of a double-stranded nucleic acid structure as describedherein, whereby two siRNA sequences selected from Tables A-C andpreferably from Table A, ID Nos: 14, 22, 23, 25, 27, 39, 41, 42, 49 and50 are covalently or non-covalently bound or joined by a linker to forma tandem siRNA molecule. Such tandem siRNA molecules comprising twosiRNA sequences would typically be of 38-150 nucleotides in length, morepreferably 38 or 40-60 nucleotides in length, and longer accordingly ifmore than two siRNA sequences are included in the tandem molecule. Alonger tandem molecule comprised of two or more longer sequences whichencode siRNA produced via internal cellular processing, e.g., longdsRNAs, is also envisaged, as is a tandem molecule encoding two or moreshRNAs. Such tandem molecules are also considered to be a part of thepresent invention, and further information concerning them is givenbelow.

Said combined or tandem structures have the advantage that toxicityand/or off-target effects of each siRNA are minimized, while theefficacy is increased.

In particular the siRNA used in the Examples has been such modified suchthat a 2′ O-Me group was present on the first, third, fifth, seventh,ninth, eleventh, thirteenth, fifteenth, seventeenth and nineteenthnucleotide of the antisense strand, whereby the very same modification,i.e. a 2′-O-Me group was present at the second, fourth, sixth, eighth,tenth, twelfth, fourteenth, sixteenth and eighteenth nucleotide of thesense strand. Additionally, it is to be noted that the in case of theseparticular nucleic acids according to the present invention the firststretch is identical to the first strand and the second stretch isidentical to the second strand and these nucleic acids are also bluntended. The siRNA was phosphorylated but it is envisaged that anun-phosphorylated version may be simpler to prepare in large scale andsaid un-phosphorylated REDD14, termed REDD-14NP, was found to be just asbiologically active as REDD-14 in a CNV model (see Example 6). Thesequence of this siRNA used in the experiments in Examples 6-8 is thatof REDD14, i.e., the sequence having internal reference No. 14 (seeTable A).

The terminal region of the oligonucleotide refers to bases 1-4 and/or16-19 in the 19-mer sequences (Tables A and B below) and to bases 1-4and/or 18-21 in the 21-mer sequences (Table C below).

Additionally, the siRNAs used in the present invention areoligoribonucleotides wherein one strand comprises consecutivenucleotides having, from 5′ to 3′, the sequence set forth SEQ ID NOS:53-102 or SEQ ID NOS: 175-246 or SEQ ID NOS: 296-344 (antisense strands)or a homolog thereof wherein in up to 2 of the nucleotides in eachterminal region a base is altered. Thus, in particular aspects theoligonucleotide comprises a double-stranded structure, whereby suchdouble-stranded structure comprises a first strand and a second strand,whereby the first strand comprises a first stretch of contiguousnucleotides and the second strand comprises a second stretch ofcontiguous nucleotides, whereby the first stretch is eithercomplementary or identical to a nucleic acid sequence coding for geneRTP801 and whereby the second stretch is either identical orcomplementary to a nucleic acid sequence coding for RTP801. Said firststretch comprises at least 14 nucleotides, preferably at least 18nucleotides and even more preferably 19 nucleotides or even at least 21nucleotides. In an embodiment the first stretch comprises from about 14to 40 nucleotides, preferably about 18 to 30 nucleotides, morepreferably from about 19 to 27 nucleotides and most preferably fromabout 19 to 23 nucleotides. In an embodiment the second stretchcomprises from about 14 to 40 nucleotides, preferably about 18 to 30nucleotides, more preferably from about 19 to 27 nucleotides and mostpreferably from about 19 to 23 nucleotides or even about 19 to 21nucleotides. In an embodiment the first nucleotide of the first stretchcorresponds to a nucleotide of the nucleic acid sequence coding forRTP801, whereby the last nucleotide of the first stretch corresponds toa nucleotide of the nucleic acid sequence coding for RTP801. In anembodiment the first stretch comprises a sequence of at least 14contiguous nucleotides of an oligonucleotide, whereby sucholigonucleotide is selected from the group comprising SEQ. ID. Nos.3-344, preferably from the group comprising the oligoribonucleotides ofhaving the sequence of any of the serial numbers 14, 22, 23, 25, 27, 39,41, 42, 49 and 50 in Table A. Additionally specifications of the siRNAmolecules used in the present invention may provide anoligoribonucleotide wherein the dinucleotide dTdT is covalently attachedto the 3′ terminus, and/or in at least one nucleotide a sugar residue ismodified, possibly with a modification comprising a 2′-O-methylmodification. Further, the 2′ OH group may be replaced by a group ormoiety selected from the group comprising —H—OCH₃, —OCH₂CH₃, —OCH₂CH₂CH₃, —NH₂, and F. Further, the preferable compounds of the presentinvention as disclosed above may be phosphorylated ornon-phosphorylated.

Additionally, the siRNA used in the present invention may be anoligoribonucleotide wherein in alternating nucleotides modified sugarsare located in both strands. Particularly, the oligoribonucleotide maycomprise one of the sense strands wherein the sugar is unmodified in theterminal 5′ and 3′ nucleotides, or one of the antisense strands whereinthe sugar is modified in the terminal 5′ and 3′ nucleotides.

Additionally, further nucleic acids to be used in the present inventioncomprise at least 14 contiguous nucleotides of any one of the SEQ. ID.NO. 3 to 344, and more preferably 14 contiguous nucleotide base pairs atany end of the double-stranded structure comprised of the first stretchand second stretch as described above. It will be understood by oneskilled in the art that given the potential length of the nucleic acidaccording to the present invention and particularly of the individualstretches forming such nucleic acid according to the present invention,some shifts relative to the coding sequence of the RTP801 gene asdetailed in SEQ ID NO:1 to each side is possible, whereby such shiftscan be up to 1, 2, 3, 4, 5 and 6 nucleotides in both directions, andwhereby the thus generated double-stranded nucleic acid molecules shallalso be within the present invention.

An additional aspect of the present invention concerns a functionalnucleic acid comprising a double-stranded structure, whereby suchdouble-stranded structure comprises

-   -   a first strand and a second strand, whereby    -   the first strand comprises a first stretch of contiguous        nucleotides and the second strand comprises a second stretch of        contiguous nucleotides, whereby    -   the first stretch is either complementary or identical to a        nucleic acid sequence coding for RTP801 and whereby the second        stretch is either identical or complementary to a nucleic acid        sequence coding for RTP801.

In an embodiment the nucleic acid is down-regulating RTP801, whereby thedown-regulation of RTP801 is selected from the group comprisingdown-regulation of RTP801 function, down-regulation of RTP801 proteinand down-regulation of RTP801 mRNA expression.

In an embodiment the first stretch comprises at least 14 nucleotides,preferably at least 18 nucleotides and even more preferably 19nucleotides.

In an embodiment the first stretch comprises from about 14 to 40nucleotides, preferably about 18 to 30 nucleotides, more preferably fromabout 19 to 27 nucleotides and most preferably from about 19 to 23nucleotides.

In an embodiment the second stretch comprises from about 14 to 40nucleotides, preferably about 18 to 30 nucleotides, more preferably fromabout 19 to 27 nucleotides and most preferably from about 19 to 23nucleotides.

In an embodiment the first nucleotide of the first stretch correspondsto a nucleotide of the nucleic acid sequence coding for RTP801, wherebythe last nucleotide of the first stretch corresponds to a nucleotide ofthe nucleic acid sequence coding for RTP801.

In an embodiment one stretch comprises a sequence of at least 14contiguous nucleotides of a nucleic acid sequence, whereby such nucleicacid sequence is selected from the sequences disclosed in Tables A-C,preferably from the group comprising SEQ. ID. NOs 53, 66, 67, 72, 73,74, 75, 76, 77, 91, 92, 93, 94, 96, 101 and 102, more preferablyselected from the group comprising SEQ. ID. Nos 66, 75, 79, 91, 94, 101and 102, and most preferably selected from the group comprising SEQ. ID.Nos 66, 74, 75 and 79.

In an embodiment the other stretch comprises a sequence of at least 14contiguous nucleotides of a nucleic acid sequence, whereby such nucleicacid sequence is selected from the sequences disclosed in Tables A-C,preferably from the group comprising SEQ. ID. NOs. 3, 16, 22, 23, 24,25, 26, 27, 29, 41, 42, 43, 44, 45, 46, 51 and 52, more preferablyselected from the group comprising SEQ. ID. Nos 16, 24, 25, 29, 41, 44,51, and 52, and most preferably selected from the group comprising SEQ.ID. Nos 16, 24, 25 and 29.

In an embodiment

-   -   the first stretch has a sequence according to SEQ. ID. NO. 53        and the second stretch has a sequence according to SEQ. ID. NO.        3;    -   the first stretch has a sequence according to SEQ. ID. NO. 66        and the second stretch has a sequence according to SEQ. ID. NO.        16;    -   the first stretch has a sequence according to SEQ. ID. NO. 67        and the second stretch has a sequence according to SEQ. ID. NO.        17;    -   the first stretch has a sequence according to SEQ. ID. NO. 72        and the second stretch has a sequence according to SEQ. ID. NO.        22;    -   the first stretch has a sequence according to SEQ. ID. NO. 73        and the second stretch has a sequence according to SEQ. ID. NO.        23;    -   the first stretch has a sequence according to SEQ. ID. NO. 74        and the second stretch has a sequence according to SEQ. ID. NO.        24;    -   the first stretch has a sequence according to SEQ. ID. NO. 75        and the second stretch has a sequence according to SEQ. ID. NO.        25;    -   the first stretch has a sequence according to SEQ. ID. NO. 76        and the second stretch has a sequence according to SEQ. ID. NO.        26;    -   the first stretch has a sequence according to SEQ. ID. NO. 77        and the second stretch has a sequence according to SEQ. ID. NO.        27;    -   the first stretch has a sequence according to SEQ. ID. NO. 79        and the second stretch has a sequence according to SEQ. ID. NO.        29;    -   the first stretch has a sequence according to SEQ. ID. NO. 91        and the second stretch has a sequence according to SEQ. ID. NO.        41;    -   the first stretch has a sequence according to SEQ. ID. NO. 92        and the second stretch has a sequence according to SEQ. ID. NO.        42;    -   the first stretch has a sequence according to SEQ. ID. NO. 93        and the second stretch has a sequence according to SEQ. ID. NO.        43;    -   the first stretch has a sequence according to SEQ. ID. NO. 94        and the second stretch has a sequence according to SEQ. ID. NO.        44;    -   the first stretch has a sequence according to SEQ. ID. NO. 95        and the second stretch has a sequence according to SEQ. ID. NO.        45;    -   the first stretch has a sequence according to SEQ. ID. NO. 96        and the second stretch has a sequence according to SEQ. ID. NO.        46;    -   the first stretch has a sequence according to SEQ. ID. NO. 101        and the second stretch has a sequence according to SEQ. ID. NO.        51; and    -   the first stretch has a sequence according to SEQ. ID. NO. 102        and the second stretch has a sequence according to SEQ. ID. NO.        52.

In an embodiment the first stretch has a nucleic acid sequence which isselected from the group comprising SEQ. ID. NO. 53, 66, 72, 73, 74, 75,76, 77, 79, 91, 92, 93, 94, 95, 96, 101 and 102.

It is to be understood that while the terms “first” and “second” stretchare used in connection with the nucleic acids of the present invention,they are used for the sake of convenience alone, and any nucleic acidmolecule of the invention that is described as having a first stretchwith the sequence X and a second stretch with the sequence Y, could alsoequally be described as having a first stretch with the sequence Y and asecond stretch with the sequence X, so long as it is understood that onestrech is comprised in the antisense strand, which must be antisense toa portion of the coding sequence of the RTP801 gene, and the otherstrech is comprised in the sense strand, which must be complimentary(although not 100% complimentary) to the antisense strand, all accordingo the definitions and specifications presented herein.

In an embodiment the first and/or the second strand comprises at leastone overhang nucleotide at the 3′ end which is complementary oridentical to the corresponding nucleotide of a nucleic acid sequencecoding for RTP801.

In an embodiment the first and/or the second strand comprises from 1 to15 overhang nucleotides at the 3′ end, preferably the first and/or thesecond strand comprises from 1 to 10 overhang nucleotides at the 3′ end,more preferably the first and/or the second strand comprises from 1 to 5overhang nucleotides at the 3′ end, and most preferably the first and/orthe second strand comprises from 1 to 2 overhang nucleotides at the 3′end.

In an embodiment the first strand and/or the second strand comprises atleast one overhang nucleotide which is different from the correspondingnucleotide of the nucleic acid sequence coding for RTP801.

In an embodiment the first strand comprises two overhang nucleotideswhich are different form the corresponding nucleotide of a nucleic acidsequence coding for RTP801.

In an embodiment the first strand consists of the first stretch only.

In an embodiment the second strand consists of the second stretch only.

In an embodiment the first stretch and/or the first strand comprise(s)ribonucleotides.

In an embodiment the second stretch and/or the second strand comprise(s)ribonucleotides.

In an embodiment the first stretch and/or the second strand consist(s)of ribonucleotides.

In an embodiment some or all of the nucleotides are modified.

In a preferred embodiment such modification is related to the nucleobasemoiety of the nucleotide, to the sugar moiety of the nucleotide and/orto the phosphate moiety of the nucleotide.

In a more preferred embodiment the modification is a modification of asugar moiety and the modification is a modification at the 2′ position,whereby the 2′ OH group is replaced by a group or moiety selected fromthe group comprising —H—OCH₃, —OCH₂CH₃, —OCH₂CH₂ CH₃, —NH₂, and —F.

In an embodiment the modification is a modification of the nucleobasemoiety and the modification or modified nucleobase is selected from thegroup comprising inosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl, 2-propyl and other alkyladenines, 5-halo uracil,5-halocytosine, 5-halo cytosine, 6-azacytosine, 6-aza thymine,pseudo-uracil, 4-thiouracil, 8-halo adenine, 8-aminoadenine, 8-thioladenine, 8-thiolalkyl adenines, 8-hydroxyl adenine and other8-substituted adenines, 8-halo guanines, 8-amino guanine, 8-thiolguanine, 8-thioalkyl guanine, 8-hydroxylguanine and other substitutedguanines, other aza- and deaza adenines, other aza- and deaza guanines,5-trifluoromethyl uracil and 5-trifluoro cytosine.

In an embodiment the modification is a modification of the phosphatemoiety, whereby the modified phosphate moiety is selected from the groupcomprising phosphothioate.

In an embodiment the first stretch and/or the second stretch comprises aplurality of groups of modified nucleotides having a modification at the2′ position, whereby within the stretch each group of modifiednucleotides is flanked on one or both sides by a flanking group ofnucleotides, whereby the flanking nucleotides forming the flanking groupof nucleotides are either a non-modified nucleotide or a nucleotidehaving a modification different from the modification of the modifiednucleotides.

In a preferred embodiment the first stretch and/or the second stretchconsists of ribonucleotides.

In a more preferred embodiment the first and the second stretch comprisea plurality of groups of modified nucleotides.

In an embodiment the first stretch comprises said plurality of groups ofmodified nucleotides.

In an embodiment the second stretch comprises said plurality of groupsof modified nucleotides.

In an embodiment each group of modified nucleotides and/or each group offlanking nucleotides comprises a number of nucleotides, whereby thenumber is selected from the group comprising one nucleotide to tennucleotides.

In an embodiment the first stretch comprises a first pattern of modifiednucleotides and the second stretch comprises a second pattern ofmodified nucleotides.

In an embodiment the first pattern is the same pattern as the secondpattern.

In another embodiment the first pattern aligns with the second pattern.

In a preferred embodiment the first pattern is shifted by one or morenucleotides relative to the second pattern.

In an embodiment each of the groups of modified nucleotides consists ofone modified nucleotides and each of the groups of flanking nucleotidesconsists of one non-modified nucleotide or a nucleotide having amodification which is different from the modification of the modifiednucleotides.

In a preferred embodiment the modified nucleotide has a —OMe group atthe 2′ position.

In a preferred embodiment the flanking nucleotide is a ribonucleotidewhich has a 2′ OH group.

In an embodiment the first stretch starts with a modified nucleotide atthe 5′ end and every other nucleotide of the stretch is also a modifiednucleotide, whereas a second nucleotide starting from the 5′ end andevery other nucleotide is a non-modified nucleotide or a nucleotidehaving a modification which is different from the modification of themodified nucleotide(s).

In an embodiment the first stretch is in antisense orientation to thenucleic acid sequence coding for RTP801.

An additional aspect of the present invention related to apharmaceutical composition comprising a nucleic acid according to thefirst aspect of the present invention and/or a vector according to thesecond aspect of the present invention and preferably a pharmaceuticallyacceptable carrier; said composition optionally being for systemic orfor local administration.

In an embodiment the composition is for the treatment of a disease,whereby the disease is selected from the group comprising tumordiseases.

In an additional aspect, the problem underlying the present invention issolved by a method for the prevention and/or treatment of a patient inneed of such prevention and/or treatment comprising the administrationof a nucleic acid according to the present invention and/or vectoraccording to the present invention and/or a pharmaceutical compositionaccording to the present invention.

In an additional embodiment, a nucleic acid according to the presentinvention and/or a vector according to the present invention are usedfor the manufacture of a medicament. The medicament may be for theprevention and/or treatment of a disease, whereby such disease isselected from the group comprising tumor diseases. The tumor disease maybe selected from the group comprising solid tumors, metastatic tumorsincluding PTEN negative tumors, tumors which are drug resistant andtumors where RTP801 inhibition can be used for sensitization. Further,the tumor disease may be a late-stage tumor disease, or may involvecells which are tumor suppressor negative; said tumor suppressor may bePTEN.

An additional aspect of the present invention is solved by a method fordesigning or screening a nucleic acid which is suitable to down-regulateRTP801, comprising the following steps:

-   -   a) designing or screening a nucleic acid which is suitable to        down-regulate RTP801;    -   b) assessing defect of a nucleic acid according to any of the        above aspects of the present invention; and    -   c) comparing the effect of the nucleic acid of step a) with the        effect of the nucleic acid of step b).

In an embodiment the effect is the down-regulation of RTP801.

An additional aspect of the present invention is the use of a nucleicacid according to the present invention as a sensitizer, particularly asa sensitizer in the treatment of a disease, whereby such disease ispreferably selected from the group comprising tumor and moreparticularly tumors which are resistant to a treatment usingchemotherapeutics and/or radiotherapeutics. Additional diseases forwhich a nucleic acid of the present invention can serve as a sensitizerare disclosed herein.

This application discloses that a nucleic acid comprising adouble-stranded structure which is specific for RTP801 is a suitablemeans of inhibiting angiogenesis/growth of vasculature and vascularleakage, (both from the existing vasculature and from growingvasculature). Additionally, this application discloses (without beingbound by theory) that RTP801 being a stress-inducible protein (inducedby hypoxia, oxidative stress, thermal stress, ER stress) is a factoracting in fine-tuning of cell response to energy disbalance. Thusinhibition of RTP801 by such double-stranded nucleic acid is suitablefor treatment of any disease where cells should be rescued fromapoptosis due to stressful conditions (e.g. diseases accompanied bydeath of normal cells) or where cells adapted to stressful conditionsdue to changes in RTP801 expression, should be killed (e.g. tumorcells). In the latter case, upon inhibiting RTP801 through suchdouble-stranded nucleic acid, this survival factor with anti-apoptoticfunction in hypoxic cells, more particularly hypoxic cancer cells, ismade ineffective thus allowing the cells devoid of RTP801-mediatedprotection to be driven into apoptosis. This can additionally occur whenother apoptosis promoting factors are present Such other apoptosispromoting factors include, among others, chemotherapy and radiationtherapy. In other words, the double-stranded nucleic acid according tothe present invention may be effective alone in cancer treatment(monotherapy) and also as a supplementary therapy.

Such double-stranded structure comprises a first strand and a secondstrand, whereby the first strand comprises a first stretch of contiguousnucleotides and the second strand comprises a second stretch ofcontiguous nucleotides, whereby the first stretch is eithercomplementary or identical to a nucleic acid sequence coding for RTP801and whereby the second stretch is either identical or complementary to anucleic acid sequence coding for RTP801. By particularly using RTP801 asa target for such kind of double-stranded nucleic acid, it is thus alsopossible to immediately address a target in the cascade involved in thegrowth and development of vasculature and angiogenesis, respectively,and thus in a different way compared to the pathway used by VEGFinhibitors such as VEGF antibodies. Without wishing to be bound by anytheory, the present inventors assume that the nucleic acid according tothe present invention may exert its function in those cells whichprovide for a background which is involved in or observed in connectionwith any disease where undesired, particularly hypoxia inducedangiogenesis and/or growth or development of vasculature occurs. Thisunderstanding is supported by the finding that RTP801 knock-out mice donot exhibit any phenotype different from wildtype mice under non-hypoxicconditions. Only upon induction of hypoxia as observed in a diseasedcondition such as, e.g., tumor growth, the RTP801 related knock-outresults in a pathology similar to the one observed in humans sufferingfrom this kind of disease.

It is to be understood that the nucleic acid according to the presentinvention is preferably a functional nucleic acid. As used herein, theterm functional nucleic acid preferably means a nucleic acid thefunction of which is different from being active in the cell as atemplate for the transcription of any hnRNA, mRNA, or any othertranscription product, whereby either said hnRNA, mRNA or any othertranscription product, respectively, or the nucleic acid according tothe present invention is subject to a translation process, preferably acellular translation process, resulting in a biologically active RTP801protein. It is to be acknowledged that a functional nucleic acid aspreferably used herein is capable of reducing the expression of a targetnucleic acid. More preferably, such reduction is based on apost-transcriptional gene silencing process of the target nucleic acid.Even more preferably such reduction is based on RNA interference. A mostpreferred form of the functional nucleic acid is an siRNA molecule orany further molecule having the same effect as an siRNA molecule. Suchfurther molecule is selected from the group comprising siRNAs, syntheticsiRNAs, shRNAs and synthetic shRNAs. As used herein siRNAs mayadditionally comprise expression vector derived siRNAs, whereby theexpression vector is in a preferred embodiment a virus such asAdenoviruses, Adenoassociated viruses, Herpes viruses and Lentiviruses.As used herein shRNA preferably means short hairpin RNAs. Such shRNA canbe made synthetically or can be generated using vector encodedexpression systems, preferably using RNA polymerase III promoters. Inconnection therewith it is to be acknowledged that the functionalnucleic acid according to the present invention is directed to RTP801which is also preferably referred to herein as the target and thenucleic acid coding for said target as the target nucleic acid.

As preferably used herein, the double-stranded structure of the nucleicacid according to the present invention comprises any double-strandedstructure, whereby such double-stranded structure is preferablygenerated by the first stretch and the second stretch provided by thenucleic acid having the basic design. The double-stranded structure maycomprise one or several mismatches. Such double-stranded structure isformed by Watson-Crick-base pairing and/or Hoogsteen base pairing and/orsimilar base pairing mechanisms. Based on the basic design of thenucleic acid according to the present invention it is preferred that onestretch, is in antisense orientation to a nucleic acid sequence codingfor RTP801 or a part thereof, whereas the other stretch is in the senseorientation to a nucleic acid sequence coding for RTP801 or a partthereof. Because of this, one stretch is complementary to a nucleic acidsequence coding for RTP801 or a part thereof, and the other stretch isidentical to a nucleic acid sequence coding for RTP801 or a partthereof. In connection therewith it is to be acknowledged that the termidentical, of course, means also partially identical, whereby theidentity, expressed as homology, is at least 80%, preferably 90%, morepreferably 95%, 96%, 97%, 98%, 99% or 100%. Similar to the definition ofidentity, complementarity can be defined in terms of homology, wherebysuch homology is of the same range as the identity if the complementarystrand would be translated into the identical strand according toWatson-Crick base pairing rules. In an alternative embodiment, onestretch is identical to a nucleic acid sequence coding for RTP801 or apart thereof and the other stretch is complementary to a nucleic acidsequence coding for RTP801 or a part thereof.

In a preferred embodiment, the nucleic acid according to the presentinvention is down-regulating RTP801 function. Down-regulation of RTP801function preferably happens by reduction in the level of expression atthe protein level and/or the mRNA level, whereby such reduced level ofexpression, preferably at the protein level, can be as little as 5% andbe as high as 100%, with reference to an expression under conditionswhere the nucleic acid according to the present invention is notadministered or is not functionally active. Such conditions arepreferably the conditions of or as present in an expression system,preferably an expression system for RTP801. Such expression system ispreferably a translation system which can be an in vitro translationsystem, more preferably a cell, organ and/or organism. It is morepreferred that the organism is a multicellular organism, more preferablya mammal, whereby such mammal is preferably selected from the groupcomprising man, monkey, mouse, rat, guinea pig, rabbit, cat, dog, sheep,cow, horse, cattle and pig. In connection with the down-regulation it isto be acknowledged that said down-regulation may be a function of time,i.e. the down-regulation effect is not necessarily observed immediatelyupon administration or functional activation of the nucleic acidsaccording to the present invention, but may be deferred in time as wellas in space, i.e. in various cells, tissues and/or organs. Suchdeferment may range from 5%-100%, preferably 10 to 50%. It will beacknowledged by the ones skilled in the art that a 5% reduction for alonger time period might be as effective as a 100% reduction over ashorter time period. It will also be acknowledged by the ones skilled inthe art that such deferment strongly depends on the particularfunctional nucleic acid actually used, as well as on the target cellpopulation and thus, ultimately, on the disease to be treated and/orprevented according to the technical teaching of the presentapplication. Insofar, a 5% reduction over a longer time period might beas effective as 100% reduction over a shorter time period. It will alsobe acknowledged by the ones skilled in the art that the deferment canoccur at any level as outlined above, i.e. a deferment in function,whereby such function is any function exhibited by RTP801, a defermentin protein expression or a deferment at mRNA expression level.

In a preferred embodiment the first stretch comprises at least 14nucleotides, preferably 14 contiguous nucleotides. It will beacknowledged by the one skilled in the art that the first stretch shouldhave a length which is suitable to allow for specifically addressing anucleic acid sequence coding for RTP801 and more specifically thenucleic acid coding for RTP801 as present in the translation systemwhere the expression of RTP801 is to be reduced. Again without wishingto be bound by any theory or any mode of action of the nucleic acidaccording to the present invention, it seems that there is aninteraction between the nucleic acid according to the present inventionand the nucleic acid sequence coding for RTP801, preferably at thetranscript level, i.e. upon generation of an mRNA from the respectivenucleic acid sequence coding for RTP801. Due to the likelihood of anysequence of the nucleic acid according to the present invention beingidentical to or complementary to a sequence contained in the genome ortranscriptome of the translation system, the length of the first stretchshould thus be as long as to make sure that, under the assumption thatsome kind of base pairing between the nucleic acid coding for RTP801 andone of the strands of the nucleic acid according to the presentinvention actually occurs, only the sequence coding for RTP801 but noother coding sequence, preferably no other essential coding sequence, ofthe genome or the transcriptome is addressed for or by such basepairing. By this length, the occurrence of off-target effects can bereduced and preferably eliminated. To increase the stringency of thiskind of specifically addressing RTP801 and the nucleic acid sequencecoding therefor, the first stretch preferably has a length of at least18 or 19 nucleotides. The upper limit for the length of the firststretch is preferably less than 50 nucleotides, however, the length canbe significantly longer and can comprise 100, 200 or even 500nucleotides or any length in-between. Apart from this, one skilled inthe art will prefer to have a rather short first stretch, particularlyin case the nucleic acid according to the present invention ischemically synthesised as the shorter the sequence is, the less time andmaterial consuming the synthesis thereof will be and the lower will bethe rate at which incorrect nucleotides are inserted into the respectivesequence. Another factor which is to be taken into consideration inconnection with fixing the length of the first stretch is the fact that,typically at a length beyond 50 or more nucleotides, an unspecificinterferon response may be observed. It depends on the particularcondition to be treated whether this kind of unspecific interferonresponse is to be tolerated or not. For example, an interferon responsecould be tolerated if the interferon response and/or the expression ofthe interferon genes can be limited to the pathogenic cells.

In view of this, more preferred lengths of the first stretch are fromabout 14 to 40 nucleotides, 18 to 30 nucleotides, 19 to 27 nucleotides,21 to 25 nucleotides and 19 to 23 nucleotides.

The same considerations as outlined above for the first stretch areapplicable to the second stretch which may thus comprise any length asdescribed herein in connection with the first stretch. It is also withinthe present invention that the length of the first stretch is differentfrom the length of the second stretch, however, it is preferred thatboth stretches have the same length.

According to the basic design of the nucleic acid, the first stretch andsecond stretch are parts of the first strand and second strand,respectively, of the nucleic acid according to the present invention. Itwill be acknowledged that at either end, i.e. at the 5′ end as well asthe 3′ end the first strand and/or second strand may comprise one orseveral nucleotides, preferably additional nucleotides, at anycombination.

In connection therewith it is to be acknowledged that those nucleotidesof the individual strand going beyond the end(s) of the stretchcorresponding to the respective strand can be used to further contributeto the complementarity and identity, respectively, of the stretch andthus to the specific addressing of the nucleic acid sequence coding forRTP801.

It will be acknowledged that, basically, based on the technical teachingprovided herein, the nucleic acid according to the present invention canaddress any part of the nucleic acid sequence coding for RTP801,preferably coding for RTP801 in the translation system where theexpression of RTP801 is to be reduced. Insofar, the present inventioncomprises any nucleic acid having the characteristics as defined herein,whereby the complementary and identical strands and stretches of thenucleic acid according to the present invention can basically start fromany nucleotide of the nucleic acid sequence coding for RTP801.Accordingly, under the proviso that the first stretch of the nucleicacid according to the present invention is complementary to the nucleicacid sequence coding for RTP801, i.e. is the antisense strand thereof oris in antisense orientation thereto, the first nucleotide of saidstretch, i.e. the most 5′ terminal nucleotide corresponds, i.e. alignsto the last nucleotide of the sequence coding for RTP801 at the 3′ end.In a further embodiment such most 5′ terminal nucleotide corresponds tothe penultimate nucleotide of the nucleic acid coding for RTP801 and soon until the last position is reached which, given the length of theantisense stretch, still allows that the antisense strand of the nucleicacid according to the present invention is complementary to the nucleicacid sequence coding for RTP801. Insofar, any nucleic acid according tothe present invention is within the present invention which could begenerated by scanning the nucleic acid sequence coding for RTP801starting from the most 5′ terminal nucleotide thereof and laying overthe basic design of the nucleic acid according to the present inventionand realising the characteristics for such nucleic acid according to thepresent invention. The same considerations are applicable to theembodiments disclosed herein where the complementarity and identity ofthe nucleic acid according to the present invention is not only providedby the first stretch and second stretch, respectively, but suchcomplementarity and identity also involves one or more nucleotidesbeyond the first stretch and second stretch, respectively, then beingpart of the first strand and second strand, respectively.

Of the various nucleic acids according to the present invention asdisclosed herein, those with internal reference numbers 14, 22, 23, 25,27, 39, 41, 42, 49 and 50 (see Table A) are particularly preferred. Inconnection therewith it is to be noted that those nucleic acidsaccording to the present invention which can be used in both human andan animal model such as rat and/or mouse are particularly useful. Thesurprising advantage of these particular nucleic acids according to thepresent invention resides in the fact that they are effective both inhuman and in an animal model which means that the test results obtainedin the animal model can be immediately transferred from the animal modelto the human being and more particularly without the necessity to makeany changes to the human sequence which would otherwise become necessaryin case the nucleic acid according to the present invention was designedsuch as to comprise (a) sequence(s) which differ(s) between the species,more particularly the species used for animal model testing and man asthe ultimate preferred organisms or patient. It is further preferredthat these nucleic acids have a modification pattern as also describedin the examples.

However, it is also within the present invention that any of thesequences according to SEQ. ID. NOs. 3, 16-17, 22-27, 29, 41-46, 51-53,66-67, 72-77, 79, 91-96 and 101-102 and respective combinationsresulting in the nucleic acid molecules according to the presentinvention having internal reference numbers 14, 22, 23, 25, 27, 39, 41,42, 49 and 50, is only partially contained in a further nucleic acidaccording to the present invention. Preferably, the further nucleicacids according to the present invention comprise at least 14 contiguousnucleotides of the SEQ. ID. NO.s 3, 16-17, 22-27, 29, 41-46, 51-53,66-67, 72-77, 79, 91-96 and 101-102, and more preferably 14 contiguousnucleotide base pairs at any end of the double-stranded structurecomprised of the first stretch and second stretch as outlined in thepreceding table. It will be understood by the ones skilled in the artthat given the potential length of the nucleic acid according to thepresent invention and particularly of the individual stretches formingsuch nucleic acid according to the present invention, some shiftsrelative to the coding sequence of RTP801 to each side is possible,whereby such shifts can be up to 1, 2, 3, 4, 5 and 6 nucleotides in bothdirections, and whereby the thus generated double-stranded nucleic acidmolecules shall also be within the present invention.

In a preferred embodiment of the present invention the first stretch andthe first strand have the same length. Likewise it is preferred that thesecond strand has the same length as the second stretch, whereby it iseven more preferred that the first stretch and the second stretch havethe same length. In a still more preferred embodiment, the first strandonly comprises the first stretch and the second strand only comprisesthe second stretch. In an even more preferred embodiment neither thefirst stretch, and thus the first strand, nor the second stretch, andthus the second strand, comprise an overhang. In other words, it is alsowithin the present invention that the double-stranded nucleic acidsaccording to the present invention are blunt ended, preferably at eachend of the double-stranded structure of the nucleic acids according tothe present invention. Such blunt ended structure can be realized inconnection with any other embodiments of the nucleic acids according tothe present invention, particularly those embodiments where the nucleicacids according to the present invention have a modification pattern,more preferably a modification pattern as described herein.

In a further aspect, the nucleic acid according to the present inventionhas thus a basic design which provides for blunt ends at both ends ofthe double-stranded structure of the nucleic acid according to thepresent invention. However, it is also within the present invention thatthere is a overhang, i.e. a stretch of one or more nucleotidesprotruding from the double-stranded structure. The overhang can be, inprinciple, at the 5′ end of the antisense strand, at the 3′ end of theantisense strand, at the 5′ end of the sense strand and/or the 3′ end ofthe sense strand. It is to be noted that realising any single of saidoptions as well as any combination thereof is within the presentinvention. More preferred is a combination, whereby the overhang islocated at the 3′ end of the antisense strand and at the 3′ end of thesense strand. It is also within the present invention that the overhangis at the 5′ end of the antisense strand and at the 5′ end of the sensestrand. Furthermore it is within the present invention that the overhangis located only at the antisense strand of double-stranded structure,more preferably at the 3′ end of the antisense strand of thedouble-stranded structure.

In connection with the overhangs, it is to be noted that the overhangplus the stretch preferably form the strand and the lengths provided forthe stretches herein apply also to these embodiments. The individualoverhang can, independent of its location, consist of at least onenucleotide. However, the individual overhang can comprise as many as 10and is preferably two nucleotides long. It is within the presentinvention that the respective nucleotide(s) forming the overhang(s)is/are also complementary to the nucleic acid sequence coding for RTP801in case of the first strand being complementary to said nucleic acidsequence coding for RTP801, and the overhang being at the 3′ or 5′ endof the antisense strand, or that the overhang(s) is/are identical to thenucleic acid sequence coding for RTP801 in case the first strand isidentical to the nucleic acid sequence coding for RTP801. The sameapplies to any overhang located at the second stretch of the basicdesign of the nucleic acid according to the present invention, wherebyit is to be acknowledged that the overhang design at the second stretchcan be independent from the overhang design of the first stretch.

It is also within the present invention that the overhang formingnucleotides are neither complementary nor identical to the correspondingnucleotides of the nucleic acid sequence coding for RTP801. As usedherein, and preferably in this embodiment, “corresponding” means therespective nucleotides which follow at the 5′ end and/or the 3′ end ofthe stretch having a nucleotide counterpart on the nucleic acid codingfor RTP801.

Preferably, the first strand comprises at its 3′ end two nucleotides,preferably deoxynucleotides and more preferably two TT and/or this kindof nucleotides also at the 3′ end of the second strand, whereby morepreferably the length of the first stretch and the second stretch is 19nucleotides. The strands are thus comprised of the stretch and theoverhang. In this embodiment the double-stranded structure consists of19 base pairs and an overhang of two nucleotides at each end of 3′ endof the individual stretch.

In a preferred embodiment, the first stretch and/or the first strandcomprise(s) ribonucleotides, whereby it is particularly preferred thatthe first stretch consists in its entirety of ribonucleotides. The sameapplies to the second stretch and the second strand, respectively. Inconnection therewith, however, each and any of the nucleotides of thefirst stretch and second stretch, respectively, is modified in apreferred embodiment. The same applies to the first strand and secondstrand, respectively. Particularly the terminal nucleotides,irrespective whether they are ribonucleotides or deoxyribonucleotides,can have an OH-group which as such can be modified. Such OH-group maystem from either the sugar moiety of the nucleotide, more preferablyfrom the 5′position in case of the 5′OH-group and/or from the 3′positionin case of the 3′OH-group, or from a phosphate group attached to thesugar moiety of the respective terminal nucleotide. The phosphate groupmay in principle be attached to any OH-group of the sugar moiety of thenucleotide. Preferably, the phosphate group is attached to the5′OH-group of the sugar moiety in case of the free 5′OH-group and/or tothe 3′OH-group of the sugar moiety in case of the free 3′OH-group stillproviding what is referred to herein as free 5′ or 3′ OH-group.

As used herein with any strategy for the design of RNAi or anyembodiment of RNAi disclosed herein, the term end modification means achemical entity added to the most 5′ or 3′ nucleotide of the firstand/or second strand. Examples for such end modifications include, butare not limited to, 3′ or 5′ phosphate, inverted (deoxy) abasics, amino,fluoro, chloro, bromo, CN, CF, methoxy, imidazole, caboxylate, thioate,C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl or aralkyl,OCF₃, OCN, O-, S-, or N-alkyl; O-, S-, or N-alkenyl; SOCH₃; SO₂CH₃;ONO₂; NO₂, N₃; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino;polyalkylamino or substituted silyl, as, among others, described inEuropean patents EP 0 586 520 B1 or EP 0 618 925 B1.

As used herein, alkyl or any term comprising “alkyl” preferably meansany carbon atom chain comprising 1 to 12, preferably 1 to 6 and more,preferably 1 to 2 C atoms.

A further end modification is a biotin group. Such biotin group maypreferably be attached to either the most 5′ or the most 3′ nucleotideof the first and/or second strand or to both ends. In a more preferredembodiment the biotin group is coupled to a polypeptide or a protein. Itis also within the scope of the present invention that the polypeptideor protein is attached through any of the other aforementioned endmodifications. The polypeptide or protein may confer furthercharacteristics to the nucleic acid molecules according to the presentinvention. Among others the polypeptide or protein may act as a ligandto another molecule. If said other molecule is a receptor the receptor'sfunction and activity may be activated by the binding ligand. Thereceptor may show an internalization activity which allows an effectivetransfection of the ligand bound nucleic acid molecules according to thepresent invention. An example for the ligand to be coupled to theinventive nucleic acid molecule is VEGF and the corresponding receptoris the VEGF receptor.

Various possible embodiments of the RNAi of the present invention havingdifferent kinds of end modification(s) are presented in the followingtable 1. TABLE 1 VARIOUS EMBODIMENTS OF THE INTERFERING RIBONUCLEIC ACIDACCORDING TO THE PRESENT INVENTION 1^(st) strand/1^(st) stretch 2^(nd)strand/2nd stretch 1.) 5′-end free OH free OH 3′-end free OH free OH 2.)5′-end free OH free OH 3′-end end modification end modification 3.)5′-end free OH free OH 3′-end free OH end modification 4.) 5′-end freeOH free OH 3′-end end modification free OH 5.) 5′-end free OH endmodification 3′-end free OH free OH 6.) 5′-end free OH end modification3′-end end modification free OH 7.) 5′-end free OH end modification3′-end free OH end modification 8.) 5′-end free OH end modification3′-end end modification end modification

The various end modifications as disclosed herein are preferably locatedat the ribose moiety of a nucleotide of the nucleic acid according tothe present invention. More particularly, the end modification may beattached to or replace any of the OH-groups of the ribose moiety,including but not limited to the 2′OH, 3′OH and 5′OH position, providedthat the nucleotide thus modified is a terminal nucleotide. Invertedabasics are nucleotides, either desoxyribonucleotides or ribonucleotideswhich do not have a nucleobase moiety. This kind of compound is, amongothers, described in Sternberger, M., Schmiedeknecht, A., Kretschmer,A., Gebhardt, F., Leenders, F., Czauderna, F., Von Carlowitz, I., Engle,M., Giese, K., Beigelman, L. & Klippel, A. (2002). Antisense NucleicAcid Drug Dev, 12, 131-43

Any of the aforementioned end modifications may be used in connectionwith the various embodiments of RNAi depicted in Table 1; it is to benoted that the 5′ end modifications mentioned above are usually onlypresenti in the sense strand of the siRNA molecule

Further modifications can be related to the nucleobase moiety, the sugarmoiety or the phosphate moiety of the individual nucleotide.

Such modification of the nucleobase moiety can be such that thederivatives of adenine, guanine, cytosine and thymidine and uracil,respectively, are modified. Particularly preferred modified nucleobasesare selected from the group comprising inosine, xanthine, hypoxanthine,2-aminoadenine, 6-methyl, 2-propyl and other alkyladenines, 5-halouracil, 5-halocytosine, 5-halo cytosine, 6-azacytosine, 6-aza thymine,pseudo-uracil, 4-thiouracil, 8-halo adenine, 8-aminoadenine, 8-thioladenine, 8-thiolalkyl adenines, 8-hydroxyl adenine and other8-substituted adenines, 8-halo guanines, 8-amino guanine, 8-thiolguanine, 8-thioalkyl guanine, 8-hydroxylguanine and other substitutedguanines, other aza- and deaza adenines, other aza- and deaza guanines,5-trifluoromethyl uracil and 5-trifluoro cytosine.

In another preferred embodiment, the sugar moiety of the nucleotide ismodified, whereby such modification preferably is at the 2′ position ofthe ribose and desoxyribose moiety, respectively, of the nucleotide.More preferably, the 2′ OH group is replaced by a group or moietyselected from the group comprising amino, fluoro, alkoxy and alkyl.Preferably, alkoxy is either methoxy or ethoxy. Also preferably alkylmeans methyl, ethyl, propyl, isobutyl, butyl and isobutyl. It is evenmore preferred that, regardless of the type of modification, thenucleotide is preferably a ribonucleotide.

The modification of the phosphate moiety is preferably selected from thegroup comprising phosphothioates.

It will be acknowledged by the one skilled in the art that the nucleicacid of the present invention which consists of a multitude ofnucleotides may thus be formed by nucleotides which are linked through aphosphodiester linkage or through a phosphothioate linkage, or acombination of both along the length of the nucleotide sequence of theindividual strand and stretch, respectively.

A further form of nucleotides used may actually be siNA which is, amongothers, described in international patent application WO 03/070918.

The nucleotides forming the first stretch and first strand,respectively, of the nucleic acid according to the present invention cancomprise one or more modified nucleotides, whereby the individualmodified nucleotide has a modification which is preferably amodification as disclosed herein. In addition to the particularmodification, the modification can be or comprise some sort of label,whereby the label is selected from the group chemiluminescent labels,fluorescent labels and radio labels. These kinds of labels are known tothe one skilled in the art and, e.g., described in Ausubel et al.,Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore,Md., 1998. The thus labelled nucleic acid according to the presentinvention may be used also for diagnostic purposes or for monitoring thesite of action as well as for the staging of any treatment, preferablyrelated to any of the diseases disclosed herein.

In a preferred embodiment, the nucleic acid according to the presentinvention is modified such that the pyrimidine nucleotides in the sensestretch or strand are 2′ O-methylpyrimidine nucleotides and, eitheradditionally or alternatively, the purine nucleotides in the sensestretch or strand are 2′-deoxypurine nucleotides. In a furtherembodiment the pyrimidine nucleotides present in the sense stretch orsense strand are 2′-deoxy-2′-fluoro pyrimidine nucleotides.

In an alternative embodiment, the modification is not based on thechemistry of the nucleotide, i.e. the modification depends on whetherthe nucleotide to be modified is either a purine nucleotide or apyrimidine nucleotide, but is predominantly based on the individualnucleotide's spatial arrangement in the overall double-strandedstructure of the basic design of the nucleic acid according to thepresent invention.

More particularly, either the first strand and first stretch,respectively, or the second strand and second stretch, respectively,show a spatial pattern of modification of the nucleotides forming saidstretches and strands, respectively.

Focusing on the first stretch first, there is a pattern of groups ofmodified nucleotides and groups of non-modified nucleotides. Thesegroups of non-modified nucleotides are also referred to herein asflanking groups of nucleotides. More preferably, the pattern consists ofgroups of modified nucleotides and non-modified nucleotides. Even morepreferably, the pattern is a regular pattern and even more preferably analternating pattern along the length of the first stretch of the nucleicacid according to the present invention. The group of modifiednucleotides may either consist of one or of several nucleotides whichare modified and which are preferably nucleotides which are modified atthe 2′ position, i.e. have a modification at the sugar moiety. Morepreferably, this modification is a 2′-O-Me modification.

The group of non-modified nucleotides may either consist of one or ofseveral nucleotides which are either not modified, whereby thenot-modified nucleotides are preferably ribonucleotides, or the notmodified nucleotides are nucleotides having a modification, whereby suchmodification is different from the modification shown by the nucleotidesforming the group of modified nucleotides. Even more preferably, the notmodified nucleotides are ribonucleotides. It is to be noted that theterm not modified and non-modified nucleotide are used in aninterchangeable manner if not indicated to the contrary. The firststretch of the nucleic acid according to the present invention mayeither start with a group of modified nucleotides or start with a groupof non-modified nucleotides as defined herein. However, it is preferredthat the first stretch starts with a group of modified nucleotides. Mostpreferably, the group of modified nucleotides consists of a singlenucleotide. In connection with this embodiment the first stretch ispreferably in antisense orientation to the nucleic acid coding forRTP801. It is also within the present invention that the modification asexhibited by the nucleotides forming the group of modified nucleotidesis the same for all groups of modified nucleotides present on the firststretch. However, it is also within the present invention that somegroup of modified nucleotides have a different modification than one orseveral groups of modified nucleotides present on the first stretch.

On the second strand of the nucleic acid according to the presentinvention, a pattern as described for the first stretch can also berealised. The same characteristics as described in connection with thefirst stretch can be realized in an embodiment on the second stretch aswell, whereby it is preferred that, under the proviso that the secondstretch is in sense orientation relative to the nucleic acid sequencecoding for RTP801, the second strand of the nucleic acid according tothe present invention starts with a group of non-modified nucleotides.

The nucleic acid according to the present invention comprising adouble-stranded structure may comprise a first stretch having themodification pattern as described herein. Alternatively, thedouble-stranded nucleic acid according to the present invention maycomprise a second stretch having the modification pattern as outlinedabove. It is, however, most preferred that the double-stranded nucleicacid according to the present invention consists of a first stretch anda second stretch, whereby both the first stretch and the second stretchhave a spatial modification pattern as described herein.

It is within the present invention that the characteristics of thespatial modification pattern is the same on both stretches in terms ofsize of the groups of modified nucleotides and groups of non-modifiednucleotides and the kind of modifications actually used. Preferably, thespatial pattern of modification on the first stretch is shifted suchthat a group of modified nucleotides on the first stretch is opposing agroup of non-modified nucleotides on the second stretch and vice versa.However, it is also with the present invention that the patterns areexactly aligned, i.e. that a group of modified nucleotides on the firststretch is opposing a group of non-modified nucleotides on the secondstretch and a group of non-modified nucleotides on the first stretch isopposing a group of non-modified nucleotides on the second stretch. Itis still within the present invention that the spatial pattern ofmodification on the first stretch and the second stretch is shiftedrelative to each other so that only a first portion of a group ofmodified nucleotides on one stretch is opposing a portion of a group ofnon-modified nucleotides on the other stretch, whereas the secondportion of the group of modified nucleotides is opposing another groupof modified nucleotides. It is within the present invention that thedisclosure provided herein on the spatial modification pattern of thestretch(es) of the nucleic acid according to the present inventionapplies also to the strand(s) of the nucleic acid according to thepresent invention. However, it is preferred that the stretches of thenucleic acid comprise the spatial modification pattern and the strandscomprise such stretches and one or more overhang(s) as disclosed herein.It is particularly preferred that the overhang is a phosphate group atthe 3′ end of either the antisense strand, or the sense strand or bothstrands, whereby it is more preferred that the phosphate group is at the3′ end of both the antisense strand and the sense strand. In an evenmore preferred embodiment, the phosphate group is a phosphate group asdefined herein.

It is also within the present invention that the nucleic acid accordingto the present invention may exhibit a linker connecting the first andthe second strand. Such linker is preferably a polymer. The polymer canbe any synthetic or natural polymer. Possible synthetic linkers are,among others, PEG or a polynucleotide. Such linker is preferablydesigned such as to allow the either partial or complete folding back ofthe first stretch onto the second stretch and vice versa.

Finally, it is within the present invention that the nucleic acidaccording to the present invention is a synthetic one, a chemicallysynthesised one, an isolated one, or one derived from any naturalsources such as, for example, prepared by means of recombinanttechnology. In connection with the preparation of any nucleic acidaccording to the present invention any modification as disclosed hereincan be introduced either prior, during or subsequent to the preparationof the respective nucleic acid according to the present invention asknown to the ones skilled in the art.

The vector according to the present invention comprises a nucleic acidaccording to the present invention. Additionally, the vector may includeelements to control targeting, expression and transcription of saidnucleic acid in a cell selective manner as is known in the art. Theplasmid can include a promoter for controlling transcription of theheterologous material, i.e. the nucleic acid according to the presentinvention, and can be either a constitutive or an inducible promoter toallow selective transcription. Enhancers that may be required to obtainnecessary transcription levels can optionally be included. Enhancers aregenerally any non-translated DNA sequences which work contiguously withthe coding sequence, thus in cis, to change the basal transcriptionlevel dictated by the promoter. The expression of such constructs isknown to the one skilled in the art and may be done, e.g., by providinga respective tandem construct or by having different promoterstranscribing for the first and second strand and first and secondstretch, respectively, of the nucleic acid according to the presentinvention.

When the nucleic acid according to the present invention is manufacturedor expressed, preferably expressed in vivo, more preferably in a patientwho is in need of the nucleic acid according to the present invention,such manufacture or expression preferably uses an expression vector,preferably a mammalian expression vector. Expression vectors are knownin the art and preferably comprise plasmids, cosmids, viral expressionsystems. Preferred viral expression systems include, but are not limitedto, adenovirus, retrovirus and lentivirus.

Methods are known in the art to introduce the vectors into cells ortissues. Such methods can be found generally described in Sambrook etal., Molecular cloning: A Laboratory Manual, Cold Springs HarbourLaboratory, New York (1983, 1992), or in Ausubel et al., CurrentProtocols in Molecular Biology, John Wiley and Sons, Baltimore, Md.,1998.

Suitable methods comprise, among others, transfection, lipofection,electroporation and infection with recombinant viral vectors. Inconnection with the present invention, an additional feature of thevector is in one embodiment an expression limiting feature such as apromoter and regulatory element, respectively, that are specific for thedesired cell type thus allowing the expression of the nucleic acidsequence according to the present invention only once the background isprovided which allows the desired expression.

In a further aspect the present invention is related to a pharmaceuticalcomposition comprising a nucleic acid according to the present inventionand/or a vector according to the present invention and, optionally, apharmaceutically acceptable carrier, diluent or adjuvants or othervehicle(s). Preferably, such carrier, diluents, adjuvants and vehiclesare inert, and non-toxic. The pharmaceutical composition is in itsvarious embodiments adapted for administration in various ways. Suchadministration comprises systemic and local administration as well asoral, subcutaneous, parenteral, intravenous, intraarterial,intramuscular, intraperitonial, intranasal, and intrategral.

It will be acknowledged by the ones skilled in the art that the amountof the pharmaceutical composition and the respective nucleic acid andvector, respectively, depends on the clinical condition of theindividual patient, the site and method of administration, scheduling ofadministration, patient age, sex, bodyweight and other factors known tomedical practitioners. The pharmaceutically effective amount forpurposes of prevention and/or treatment is thus determined by suchconsiderations as are known in the medical arts. Preferably, the amountis effective to achieve improvement including but limited to improve thediseased condition or to provide for a more rapid recovery, improvementor elimination of symptoms and other indicators as are selected asappropriate measures by those skilled in the medical arts.

In a preferred embodiment, the pharmaceutical composition according tothe present invention may comprise other pharmaceutically activecompounds. Preferably, such other pharmaceutically active compounds areselected from the group comprising compounds which allow for uptakeintracellular cell delivery, compounds which allow for endosomalrelease, compounds which allow for, longer circulation time andcompounds which allow for targeting of endothelial cells or pathogeniccells. Preferred compounds for endosomal release are chloroquine, andinhibitors of ATP dependent H⁺ pumps.

The pharmaceutical composition is preferably formulated so as to providefor a single dosage administration or a multi-dosage administration.

It will be acknowledged that the pharmaceutical composition according tothe present invention can be used for any disease which involvesundesired development or growth of vasculature including angiogenesis,as well as any of the diseases and conditions described herein.Preferably, these kind of diseases are tumor diseases. Among tumordiseases, the following tumors are most preferred: endometrial cancer,colorectal carcinomas, gliomas, endometrial cancers, adenocarcinomas,endometrial hyperplasias, Cowden's syndrome, hereditary non-polyposiscolorectal carcinoma, Li-Fraumene's syndrome, breast-ovarian cancer,prostate cancer (Ali, I. U., Journal of the National Cancer Institute,Vol. 92, no. 11, Jun. 7, 2000, page 861-863), Bannayan-Zonana syndrome,LDD (Lhermitte-Duklos' syndrome) (Macleod, K., supra)hamartoma-macrocephaly diseases including Cow disease (CD) andBannayan-Ruvalcaba-Rily syndrome (BRR), mucocutaneous lesions (e.g.trichilemmonmas), macrocephaly, mental retardation, gastrointestinalharmatomas, lipomas, thyroid adenomas, fibrocystic disease of thebreast, cerebellar dysplastic gangliocytoma and breast and thyroidmalignancies (Vazquez, F., Sellers, W. R., supra).

It is to be acknowledged that any of the tumor disease to be treatedwith the pharmaceutical composition according to the present inventionis preferably a late stage tumor disease. In another embodiment, thetumor disease involves cells which are tumor suppressor negative,whereby more preferably the tumor suppressor is PTEN.

The pharmaceutical composition according to the present invention canalso be used in a method for preventing and/or treating a disease asdisclosed herein, whereby the method comprises the administration of anucleic acid according to the present invention, a vector according tothe present invention or a pharmaceutical composition or medicamentaccording to the present invention for any of the diseases describedherein.

In a further aspect, the present invention is related to a method fordesigning or screening a nucleic acid which is suitable to down-regulateRTP801, more particularly to down-regulate RTP801 function. This methodcomprises the use of a nucleic acid sequence as disclosed herein and theassessment of such nucleic acid in a suitable assay. Such assay is knownin the art and, for example, described in the example part of thisapplication. In a further step, a double-stranded nucleic acid isdesigned, preferably according to the design principles as laid downherein, which is suitable to down-regulate RTP801, preferably inconnection with a post transcriptional gene silencing mechanism such asRNA interference. Also the thus obtained, i.e. designed or screened,nucleic acid is assessed in the respective assay and the result, i.e.the effect of both the nucleic acid according to the present inventionas well as the newly designed or screened nucleic acid in such assaycompared. Preferably, the designed or screened nucleic acid is moresuitable in case it is either more stable or more effective, preferablyboth. It will be acknowledged that the method will be particularlyeffective if any of the nucleic acids according to the present inventionis used as a starting point. It is thus within the present inventionthat new nucleic acid molecules will be designed based on the principlesdisclosed herein, whereby the target sequence on the RTP801 mRNA will beslightly shifted relative to the target sequence on the RTP801 mRNA forthe corresponding nucleic acid according to the present invention.Preferably the new nucleic acid will be shifted by at least one or morenucleotides relative to the stretch on the target mRNA in either the 5′or the 3′ direction of the mRNA coding for RTP801. It is however with inthe present invention that the shift occurs in both directionssimultaneously which means that the new nucleic acid incorporates thenucleic acid according to the present invention used as a startingpoint. It is also within the present invention that the elongation ofthe nucleic acid according to the present invention and used as astarting point is biased to either the 3′ end or the 5′ end. In case ofsuch as bias either the 3′ end or the 5′ end of the new nucleic acid islonger, i.e more extended than the other end. When the new nucleic acidmolecule is generated by extending either the 3′ end of the 5′ end ofthe antisense strand and/or the sense strand, the following sequence ofsteps is typically applied. If the shift is to the 5′ end of the mRNA ofRTP801, the 3′ end of the antisense strand has to be extended by thenumber of the nucleotides by which the 5′ end of the mRNA of RTP801 isshifted. The nucleotide(s) thus to be added to the 3′ end of theantisense strand of the new nucleic acid is/are complementary to thosenucleotides following at the 5′ end of the target sequence on the RTP801mRNA used for the nucleic acid molecule according to the presentinvention used as a starting point. The same has to be done to the sensestrand. However the nucleotides to be added to the sense strand have tocorrespond, i.e. be complementary to the nucleotides newly added to the3′ end of the antisense strand which means that they have to be added tothe 5′ end of the sense strand. The latter step on the sense strand,however has to be done only to the extent that apart from the antisensestrand also the sense strand shall be shifted, which is the case inpreferred embodiments of the present invention. Although this shiftingcan be done to an extent defined by the ones skilled in the art, morepreferably the shift shall be done such that also the new nucleic acidstill contains a strech of at least 14 nucleotides, preferably 14contiguous nucleotides as exhibited by any of the nucleic acid moleculesdisclosed herein.

The synthesis of any of the nucleic acids described herein is within theskills of the one of the art. Such synthesis is, among others, describedin Beaucage S. L. and Iyer R. P., Tetrahedron 1992; 48: 2223-2311,Beaucage S. L. and Iyer R. P., Tetrahedron 1993; 49: 6123-6194 andCaruthers M. H. et. al., Methods Enzymol. 1987; 154: 287-313, thesynthesis of thioates is, among others, described in Eckstein F., Annu.Rev. Biochem. 1985; 54: 367-402, the synthesis of RNA molecules isdescribed in Sproat B., in Humana Press 2005 Edited by Herdewijn P.;Kap. 2: 17-31 and respective downstream processes are, among others,described in Pingoud A. et. al., in IRL Press 1989 Edited by Oliver R.W. A.; Kap. 7: 183-208 and Sproat B., in Humana Press 2005 Edited byHerdewijn P.; Kap. 2: 17-31 (supra).

siRNA for RTP801 can be made using methods known in the art as describedabove, based on the known sequence of RTP801 (SEQ ID NO:1), and can bemade stable by various modifications as described above. For furtherinformation, see Example 9.

Further, in relation to the methods of the present invention asdescribed herein, additional RNA molecules may be used with said methodse.g. inhibitory RNA molecules of the present invention include singlestranded oligoribonucleotides preferably comprising stretches of atleast 7-10 consecutive nucleotides present in the sequences detailed inTables A-C, said oligoribonucleotides being capable of forming [and/orcomprising] double stranded regions in particular conformations that arerecognized by intracellular complexes, leading to the degradation ofsaid oligoribonucleotides into smaller RNA molecules that are capable ofexerting inhibition of their corresponding endogenous gene, and DNAmolecules encoding such RNA molecules. The corresponding endogenous geneis preferably the 801 gene and may additionally be the VEGF gene and/orthe VEGF-R1 gene. The invention also provides a composition comprisingthe above single stranded oligoribonucleotide in a carrier, preferably apharmaceutically acceptable carrier.

Additionally, the present invention provides for combination therapy forall the conditions disclosed herein and in particular conditionsinvolving choroidal neovascularization. In said combination therapy,both the RTP801 and VEGFR genes are inhibited in order to ameliorate thesymptoms of the disease being treated. These genes may be inhibited witha combination of siRNAs or antibodies (including aptamer antibodies) orboth. The present invention therefore also provides for a novelpharmaceutical composition comprising an RTP801 inhibitor and a VEGF orVEGFR-1 inhibitor, the RTP801 inhibitor preferable being an siRNA, morepreferably an siRNA molecule detailed in Tables A-C and in particular,siRNA Nos: 14, 22, 23, 25, 27, 39, 41, 42, 49 and 50 of Table A, and theVEGF/VEGFR-1 inhibitor optionally being an antibody or aptamer. Thecombined use of said compounds (i.e., RTP801 siRNA and VEGF antibody orany other combined example disclosed herein) in the preparation of amedicament is also part of the present invention.

Thus, RTP801 siRNA such as an siRNA molecule detailed in Tables A-C andin particular, siRNA Nos: 14, 22, 23, 25, 27, 39, 41, 42, 49 and 50 ofTable A may be administered in conjunction with agents which target VEGFor VEGF receptor 1 (VEGFR1). Such agents currently exist on the marketor in various stages of approval and work through different mechanisms.Antibodies and antibody fragments such as ranibizumab (Lucentis,Genentech) attach to released VEGF to inhibit binding of VEGF to activereceptors. An aptamer which can act like a ligand/antibody (Macugen,Eyetech/Pfizer, approved recently by the FDA for wet AMD) is also apossibility. Macugen bonds with extracellular VEGF to block itsactivity. These drugs are administered locally by intravitrealinjection. Anti-VEGF siRNA based compounds (such as Acuity's Cand5inhibitor of VEGF or SIRNA's 027 inhibitor of VEGFR-1) are alsoavailable. Additionally, the small molecule aminosterol Squalamine(Genaera) which is administered systemically reportedly interferes inmultiple facets of the angiogenic process, including inhibiting VEGF andother growth factor signaling in endothelial cells.

The conjoined administration of an RTP801 inhibitor, preferably ansiRNA, and any of the above VEGF/VEGFR-1 inhibitory agents can have asynergistic effect whereby said combined treatment is more effectivethan treatment by any of these individual compositions, irrespective ofdosage in the single therapy option. This synergistic effect is alsosupported by preliminary results obtained by the Asignee, as detailed inExample 6.

RTP801i has a different mechanism of action and is potentiallysynergistic with VEGF-VEGFR inhibitors. A study in RTP801 KO miceindicates that protective phenotype in the KO mice persists in spite ofthe fact that expression of VEGF mRNA in the eye is as high as in the WTmice. Our additional preliminary data indicate that inhibition of RTP801may be synergistic with the inhibition of VEGF-VEGFR regulatory axis intreatment of retinal pathology. The inventors of the present inventionhave found in appropriate experiments that administration of siRNAagainst RTP801 in the model of AMD (see Example 6 below) leads not onlyto downregulation of RTP801 itself but also, as a consequence, toupregulation of the antiangiogenic and neuroprotective factor PEDF aswell as the downregulation of expression of MCP1, a macrophagechemoattractant protein. Thus, inhibition of RTP801 simultaneouslyconfers antiangiogenic, neuroprotective and anti-inflammatory effects.

It is to be understood that, in the context of the present invention,any of the siRNA molecules disclosed herein, or any long double-strandedRNA molecules (typically 25-500 nucleotides in length) which areprocessed by endogenous cellular complexes (such as DICER—see above) toform the siRNA molecules disclosed herein, or molecules which comprisethe siRNA molecules disclosed herein, can be employed in the treatmentof the diseases or disorders described herein.

Additional disorders which can be treated by the molecules andcompositions of the present invention include all types of choroidalneovascularization (CNV), which occurs not only in wet AMD but also inother ocular pathologies such as ocular histoplasmosis syndrome, angiodstreaks, ruptures in Bruch's membrane, myopic degeneration, oculartumors and some retinal degenerative diseases.

An additional aspect of the present invention provides for methods oftreating an apoptosis related disease. Methods for therapy of diseasesor disorders associated with uncontrolled, pathological cell growth,e.g. cancer, psoriasis, autoimmune diseases, inter alia, and methods fortherapy of diseases associated with ischemia and lack of proper bloodflow, e.g. myocardial infarction (MI) and stroke, are provided. “Cancer”or “Tumor” refers to an uncontrolled growing mass of abnormal cells.These terms include both primary tumors, which may be benign ormalignant, as well as secondary tumors, or metastases which have spreadto other sites in the body. Examples of cancer-type diseases include,inter alia: carcinoma (e.g.: breast, colon and lung), leukemia such as Bcell leukemia, lymphoma such as B-cell lymphoma, blastoma such asneuroblastoma and melanoma.

The invention also provides a composition comprising one or more of thecompounds of the invention in a carrier, preferably a pharmaceuticallyacceptable carrier. This composition may comprise a mixture of two ormore siRNAs for different genes or different siRNAs for the same gene. Acomposition comprising siRNA for the RTP801 gene and siRNA for the VEGFgene and/or the VEGF-R1 gene is envisaged.

Another compound of the invention comprises the above compound of theinvention (structure A) covalently or non-covalently bound to one ormore compounds of the invention (structure A). This compound may bedelivered in a carrier, preferably a pharmaceutically acceptablecarrier, and may be processed intracellularly by endogenous cellularcomplexes to produce one or more siRNAs of the invention. Anothercompound of the invention comprises the above compound of the invention(structure A) covalently or non-covalently bound to an siRNA for anothergene, especially the VEGF gene and/or the VEGF-R1 gene.

This invention also comprises a novel chemical entity which is an RTP801inhibitor, preferably an siRNA, chemically bound, covalently ornon-covalently, to any of the above VEGF/VEGFR-1 inhibitory agents Aparticular chemical entity envisaged is an siRNA RTP801 inhibitorcovalently bound to an antibody to VEGF or VEGF receptor-1. Methods ofproduction of such novel chemical entities are known to those skilled inthe art.

This invention also comprises a tandem double-stranded structure whichcomprises two or more siRNA sequences, which is processedintracellularly to form two or more different siRNAs, one inhibiting 801and a second inhibiting VEGF/VEGFR-1 In a related aspect, this inventionalso comprises a tandem double-stranded structure which comprises two ormore siRNA sequences, which is degraded intracellularly to form two ormore different siRNAs, both inhibiting 801.

In particular, it is envisaged that a long oligonucleotide (typicallyabout 80-500 nucleotides in length) comprising one or more stem and loopstructures, where stem regions comprise the sequences of theoligonucleotides of the invention, may be delivered in a carrier,preferably a pharmaceutically acceptable carrier, and may be processedintracellularly by endogenous cellular complexes (e.g. by DROSHA andDICER as described above) to produce one or more smaller double strandedoligonucleotides (siRNAs) which are oligonucleotides of the invention.This oligonucleotide can be termed a tandem shRNA construct. It isenvisaged that this long oligonucleotide is a single strandedoligonucleotide comprising one or more stem and loop structures, whereineach stem region comprises a sense and corresponding antisense siRNAsequence of an 801 gene. In particular, it is envisaged that thisoligonucleotide comprises sense and antisense siRNA sequences asdepicted in any one of Tables A through C. Alternatively, the tandemshRNA construct may comprise sense and corresponding antisense siRNAsequence of an 801 gene and additionally sense and correspondingantisense siRNA sequence of a different gene such as VEGF or VEGF-R1.

As mentioned herein, siRNA against RTP801 may be the main activecomponent in a pharmaceutical composition, or may be one activecomponent of a pharmaceutical composition containing two or more siRNAs(or molecules which encode or endogenously produce two or more siRNAs,be it a mixture of molecules or one or more tandem molecule whichencodes two or more siRNAs), said pharmaceutical composition furtherbeing comprised of one or more additional siRNA molecule which targetsone or more additional gene. Simultaneous inhibition of RTP801 and saidadditional gene(s) will probably have an additive or synergistic effectfor treatment of the diseases disclosed herein, according to thefollowing:

Acute Renal Failure (ARF) and other microvascular disorders: thepharmaceutical composition for treatment of ARF may be comprised of thefollowing compound combinations: 1) RTP801 siRNA and p53 siRNA dimers;2) RTP801 and Fas siRNA dimers; 3) RTP801 and Bax siRNA dimers; 4) p53and Fas siRNA dimers; 5) RTP801 and Bax siRNA dimers; 6) RTP801 and NoxasiRNA dimers; 7) RTP801 and Puma siRNA dimers; 8) RTP801 (REDD1) andRTP801L (REDD2) siRNA dimmers; 9) RTP801 siRNA, Fas siRNA and any ofRTP801L siRNA p53 siRNA, Bax siRNA, Noxa siRNA or Puma siRNA to formtrimers or polymers (i.e., tandem molecules which encode three siRNAs).

Macular degeneration (MD), diabetic retinopathy (DR), spinal cordinjury: pharmaceutical compositions for treatment of MD, DR and spinalcord injury may be comprised of the following compound combinations: 1)RTP801 siRNA combined with either of VEGF siRNA, VEGF-R1 siRNA, VEGF R2siRNA, PKCbeta siRNA, MCP1 siRNA, eNOS siRNA, KLF2 siRNA, RTP801L siRNA(either physically mixed or in a tandem molecule); 2) RTP801 siRNA incombination with two or more siRNAs of the above list (physically mixedor in a tandem molecule encodimg three siRNAs, or a combinationthereof).

COPD and respiratory disorders: the pharmaceutical composition fortreatment of respiratory disorders may be comprised of the followingcompound combinations: RTP801 siRNA combined with siRNA against one ormore of the following genes: elastases, matrix metalloproteases,phospholipases, caspases, sphingomyelinase, and ceramide synthase.

Additionally, RTP801 siRNA or any nucleic acid molecule comprising orencoding RTP801 siRNA can be linked (covalently or non-covalently) toantibodies, in order to achieve enhanced targeting for treatment of thediseases disclosed herein, according to the following:

ARF: anti-Fas antibody (preferably neutralizing antibodies).

Macular degeneration, diabetic retinopathy, spinal cord injury: anti-Fasantibody, anti-MCP1 antibody, anti-VEGFR1 and anti-VEGFR2 antibody. Theantibodies should be preferably be neutralizing antibodies.

Any molecules, such as, for example, antisense DNA molecules whichcomprise the siRNA sequences disclosed herein (with the appropriatenucleic acid modifications) are particularly desirable and may be usedin the same capacity as their corresponding siRNAs for all uses andmethods disclosed herein.

The invention also comprises a method of treating a patient sufferingfrom a disorder such as the disorders described herein comprisingadministering to the patient the above composition or compound in atherapeutically effective dose so as to thereby treat the patient.

By the term “antisense” (AS) or “antisense fragment” is meant apolynucleotide fragment (coprising either deoxyribonucleotides,ribonucleotides or a mixture of both) having inhibitory antisenseactivity, said activity causing a decrease in the expression of theendogenous genomic copy of the corresponding gene (in this case RTP801).An RTP801 AS polynucleotide is a polynucleotide which comprisesconsecutive nucleotides having a sequence of sufficient length andhomology to a sequence present within the sequence of the RTP801 geneset forth in SEQ ID NO:1 to permit hybridization of the AS to the gene.The sequence of the AS is designed to complement a target mRNA ofinterest and form an RNA:AS duplex. This duplex formation can preventprocessing, splicing, transport or translation of the relevant mRNA.Moreover, certain AS nucleotide sequences can elicit cellular RNase Hactivity when hybridized with their target mRNA, resulting in mRNAdegradation (Calabretta et al, 1996: Antisense strategies in thetreatment of leukemias. Semin Oncol. 23(1):78-87). In that case, RNase Hwill cleave the RNA component of the duplex and can potentially releasethe AS to further hybridize with additional molecules of the target RNA.An additional mode of action results from the interaction of AS withgenomic DNA to form a triple helix which can be transcriptionallyinactive. Particular AS fragments are the AS of the DNA encoding theparticular fragments of RTP801 described herein.

Many reviews have covered the main aspects of antisense (AS) technologyand its therapeutic potential (Wright & Anazodo, 1995. AntisenseMolecules and Their Potential For The Treatment Of Cancer and AIDS.Cancer J. 8:185-189.). There are reviews on the chemical (Crooke, 1995.Progress in antisense therapeutics, Hematol. Pathol. 2:59; Uhlmann andPeyman, 1990. Antisense Oligonucleotides: A New Therapeutic Principle.Chem Rev 90(4):543-584.), cellular (Wagner, 1994. Gene inhibition usingantisense oligodeoxynucleotides. Nature 372:333.) and therapeutic(Hanania, et al 1995. Recent advances in the application of gene therapyto human disease. Am. J. Med. 99:537.; Scanlon et al., 1995.Oligonucleotides-mediated modulation of mammalian gene expression. FASEBJ. 9:1288.; Gewirtz, 1993. Oligodeoxynucleotide-based therapeutics forhuman leukemias, Stem Cells Dayt. 11:96.) aspects of this technology.

Antisense intervention in the expression of specific genes can beachieved by the use of synthetic AS oligonucleotide sequences (seeLefebvre-d'Hellencourt et al, 1995. Immunomodulation by cytokineantisense oligonucleotides. Eur. Cytokine Netw. 6:7.; Agrawal, 1996.Antisense oligonucleotides: towards clinical trials, TIBTECH, 14:376.;Lev-Lehman et al., 1997. Antisense Oligomers in vitro and in vivo. InAntisense Therapeutics, A. Cohen and S. Smicek, eds (Plenum Press, NewYork)). AS oligonucleotide sequences are designed to complement a targetmRNA of interest and form an RNA:AS duplex. This duplex formation canprevent processing, splicing, transport or translation of the relevantmRNA. Moreover, certain AS nucleotide sequences can elicit cellularRNase H activity when hybridized with their target mRNA, resulting inmRNA degradation (Calabretta, et al, 1996. Antisense strategies in thetreatment of leukemias. Semin. Oncol. 23:78.). In that case, RNase Hwill cleave the RNA component of the duplex and can potentially releasethe AS to further hybridize with additional molecules of the target RNA.An additional mode of action results from the interaction of AS withgenomic DNA to form a triple helix which may be transcriptionallyinactive.

The sequence target segment for the antisense oligonucleotide isselected such that the sequence exhibits suitable energy relatedcharacteristics important for oligonucleotide duplex formation withtheir complementary templates, and shows a low potential forself-dimerization or self-complementation (Anazodo et al., 1996). Forexample, the computer program OLIGO (Primer Analysis Software, Version3.4), can be used to determine antisense sequence melting temperature,free energy properties, and to estimate potential self-dimer formationand self-complimentary properties. The program allows the determinationof a qualitative estimation of these two parameters (potentialself-dimer formation and self-complimentary) and provides an indicationof “no potential” or “some potential” or “essentially completepotential”. Using this program target segments are generally selectedthat have estimates of no potential in these parameters. However,segments can be used that have “some potential” in one of thecategories. A balance of the parameters is used in the selection as isknown in the art. Further, the oligonucleotides are also selected asneeded so that analogue substitution do not substantially affectfunction.

Phosphorothioate antisense oligonucleotides do not normally showsignificant toxicity at concentrations that are effective and exhibitsufficient pharmacodynamic half-lives in animals (Agrawal, 1996.Antisense oligonucleotides: towards clinical trials, TIBTECH, 14:376.)and are nuclease resistant. Antisense induced loss-of-functionphenotypes related with cellular development have been shown for theglial fibrillary acidic protein (GFAP), for the establishment of tectalplate formation in chick (Galileo et al., 1991. J. Cell. Biol.,112:1285.) and for the N-myc protein, responsible for the maintenance ofcellular heterogeneity in neuroectodermal cultures (ephithelial vs.neuroblastic cells, which differ in their colony forming abilities,tumorigenicity and adherence) (Rosolen et al., 1990. Cancer Res.50:6316.; Whitesell et al., 1991. Episome-generated N-myc antisense RNArestricts the differentiation potential of primitive neuroectodermalcell lines. Mol. Cell. Biol. 11:1360.). Antisense oligonucleotideinhibition of basic fibroblast growth factor (bFgF), having mitogenicand angiogenic properties, suppressed 80% of growth in glioma cells(Morrison, 1991. Suppression of basic fibroblast growth factorexpression by antisense oligonucleotides inhibits the growth oftransformed human astrocytes. J. Biol. Chem. 266:728.) in a saturableand specific manner. Being hydrophobic, antisense oligonucleotidesinteract well with phospholipid membranes (Akhter et al, 1991.Interactions of antisense DNA oligonucleotide analogs with phospholipidmembranes (liposomes) Nuc. Res. 19:5551-5559.). Following theirinteraction with the cellular plasma membrane, they are actively (orpassively) transported into living cells (Loke et al, 1989.Characterization of oligonucleotide transport into living cells. PNASUSA 86:3474.), in a saturable mechanism predicted to involve specificreceptors (Yakubov et al, 1989. PNAS USA 86:6454.).

A “ribozyme” is an RNA molecule that possesses RNA catalytic ability(see Cech for review) and cleaves a specific site in a target RNA.

In accordance with the present invention, ribozymes which cleave RTP801mRNA may be utilized as RTP801 inhibitors. This may be necessary incases where antisense therapy is limited by stoichiometricconsiderations (Sarver et al., 1990, Gene Regulation and Aids, pp.305-325). Ribozymes can then be used that will target the RTP801sequence. The number of RNA molecules that are cleaved by a ribozyme isgreater than the number predicted by stochiochemistry. (Hampel andTritz, 1989; Uhlenbeck, 1987).

Ribozymes catalyze the phosphodiester bond cleavage of RNA. Severalribozyme structural families have been identified including Group Iintrons, RNase P, the hepatitis delta virus ribozyme, hammerheadribozymes and the hairpin ribozyme originally derived from the negativestrand of the tobacco ringspot virus satellite RNA (sTRSV) (Sullivan,1994; U.S. Pat. No. 5,225,347, columns 4-5). The latter two families arederived from viroids and virusoids, in which the ribozyme is believed toseparate monomers from oligomers created during rolling circlereplication (Symons, 1989 and 1992). Hammerhead and hairpin ribozymemotifs are most commonly adapted for trans-cleavage of mRNAs for genetherapy (Sullivan, 1994). The ribozyme type utilized in the presentinvention is selected as is Known in the art. Hairpin ribozymes are nowin clinical trial and are the preferred type. In general the ribozyme isfrom 30-100 nucleotides in length. Delivery of ribozymes is similar tothat of AS fragments and/or siRNA molecules.

It will be noted that all the polynucleotides to be used in the presentinvention may undergo modifications so as to possess improvedtherapeutic properties. Modifications or analogs of nucleotides can beintroduced to improve the therapeutic properties of polynucleotides.Improved properties include increased nuclease resistance and/orincreased ability to permeate cell membranes. Nuclease resistance, whereneeded, is provided by any method known in the art that does notinterfere with biological activity of the AS polynucleotide, siRNA, cDNAand/or ribozymes as needed for the method of use and delivery (Iyer etal., 1990; Eckstein, 1985; Spitzer and Eckstein, 1988; Woolf et al.,1990; Shaw et al., 1991). Modifications that can be made tooligonucleotides in order to enhance nuclease resistance includemodifying the phophorous or oxygen heteroatom in the phosphate backbone.These include preparing methyl phosphonates, phosphorothioates,phosphorodithioates and morpholino oligomers. In one embodiment it isprovided by having phosphorothioate bonds linking between the four tosix 3′-terminus nucleotide bases. Alternatively, phosphorothioate bondslink all the nucleotide bases. Other modifications known in the art maybe used where the biological activity is retained, but the stability tonucleases is substantially increased.

All analogues of, or modifications to, a polynucleotide may be employedwith the present invention, provided that said analogue or modificationdoes not substantially affect the function of the polynucleotide. Thenucleotides can be selected from naturally occurring or syntheticmodified bases. Naturally occurring bases include adenine, guanine,cytosine, thymine and uracil. Modified bases of nucleotides includeinosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, 2-propyl andother alkyl adenines, 5-halo uracil, 5-halo cytosine, 6-aza cytosine and6-aza thymine, psuedo uracil, 4-thiuracil, 8-halo adenine,8-aminoadenine, 8-thiol adenine, 8-thiolalkyl adenines, 8-hydroxyladenine and other 8-substituted adenines, 8-halo guanines, 8-aminoguanine, 8-thiol guanine, 8-thioalkyl guanines, 8-hydroxyl guanine andother substituted guanines, other aza and deaza adenines, other aza anddeaza guanines, 5-trifluoromethyl uracil and 5-trifluoro cytosine.

In addition, analogues of polynucleotides can be prepared wherein thestructure of the nucleotide is fundamentally altered and that are bettersuited as therapeutic or experimental reagents. An example of anucleotide analogue is a peptide nucleic acid (PNA) wherein thedeoxyribose (or ribose) phosphate backbone in DNA (or RNA is replacedwith a polyamide backbone which is similar to that found in peptides.PNA analogues have been shown to be resistant to degradation by enzymesand to have extended lives in vivo and in vitro. Further, PNAs have beenshown to bind stronger to a complementary DNA sequence than a DNAmolecule. This observation is attributed to the lack of charge repulsionbetween the PNA strand and the DNA strand. Other modifications that canbe made to oligonucleotides include polymer backbones, cyclic backbones,or acyclic backbones.

The polypeptides employed in the present invention may also be modified,optionally chemically modified, in order to improve their therapeuticactivity. “Chemically modified”—when referring to the polypeptides,means a polypeptide where at least one of its amino acid residues ismodified either by natural processes, such as processing or otherpost-translational modifications, or by chemical modification techniqueswhich are well known in the art. Among the numerous known modificationstypical, but not exclusive examples include: acetylation, acylation,amidation, ADP-ribosylation, glycosylation, GPI anchor formation,covalent attachment of a lipid or lipid derivative, methylation,myristlyation, pegylation, prenylation, phosphorylation, ubiqutination,or any similar process.

Additional possible polypeptide modifications (such as those resultingfrom nucleic acid sequence alteration) include the following:

“Conservative substitution”—refers to the substitution of an amino acidin one class by an amino acid of the same class, where a class isdefined by common physicochemical amino acid side chain properties andhigh substitution frequencies in homologous polypeptides found innature, as determined, for example, by a standard Dayhoff frequencyexchange matrix or BLOSUM matrix. Six general classes of amino acid sidechains have been categorized and include: Class I (Cys); Class II (Ser,Thr, Pro, Ala, Gly); Class III (Asn, Asp, Gln, Glu); Class IV (His, Arg,Lys); Class V (Ile, Leu, Val, Met); and Class VI (Phe, Tyr, Trp). Forexample, substitution of an Asp for another class III residue such asAsn, Gln, or Glu, is a conservative substitution.

“Non-conservative substitution”—refers to the substitution of an aminoacid in one class with an amino acid from another class; for example,substitution of an Ala, a class II residue, with a class III residuesuch as Asp, Asn, Glu, or Gln.

“Deletion”—is a change in either nucleotide or amino acid sequence inwhich one or more nucleotides or amino acid residues, respectively, areabsent.

“Insertion” or “addition”—is that change in a nucleotide or amino acidsequence which has resulted in the addition of one or more nucleotidesor amino acid residues, respectively, as compared to the naturallyoccurring sequence.

“Substitution”—replacement of one or more nucleotides or amino acids bydifferent nucleotides or amino acids, respectively. As regards aminoacid sequences the substitution may be conservative or non-conservative.

In an additional embodiment of the present invention, the RTP801polypeptide or polynucleotide may be used to diagnose or detect maculardegeneration in a subject. A detection method would typically compriseassaying for RTP801 mRNA or RTP801 polypeptide in a sample derived froma subject.

“Detection”—refers to a method of detection of a disease. This term mayrefer to detection of a predisposition to a disease, or to the detectionof the severity of the disease.

By “homolog/homology”, as utilized in the present invention, is meant atleast about 70%, preferably at least about 75% homology, advantageouslyat least about 80% homology, more advantageously at least about 90%homology, even more advantageously at least about 95%, e.g., at leastabout 97%, about 98%, about 99% or even about 100% homology. Theinvention also comprehends that these polynucleotides and polypeptidescan be used in the same fashion as the herein or aforementionedpolynucleotides and polypeptides.

Alternatively or additionally, “homology”, with respect to sequences,can refer to the number of positions with identical nucleotides or aminoacid residues, divided by the number of nucleotides or amino acidresidues in the shorter of the two sequences, wherein alignment of thetwo sequences can be determined in accordance with the Wilbur and Lipmanalgorithm ((1983) Proc. Natl. Acad. Sci. USA 80:726); for instance,using a window size of 20 nucleotides, a word length of 4 nucleotides,and a gap penalty of 4, computer-assisted analysis and interpretation ofthe sequence data, including alignment, can be conveniently performedusing commercially available programs (e.g., Intelligenetics™ Suite,Intelligenetics Inc., CA). When RNA sequences are said to be similar, orto have a degree of sequence identity or homology with DNA sequences,thymidine (T) in the DNA sequence is considered equal to uracil (U) inthe RNA sequence. RNA sequences within the scope of the invention can bederived from DNA sequences or their complements, by substitutingthymidine (T) in the DNA sequence with uracil (U).

Additionally or alternatively, amino acid sequence similarity orhomology can be determined, for instance, using the BlastP program(Altschul et al., Nucl. Acids Res. 25:3389-3402) and available at NCBI.The following references provide algorithms for comparing the relativeidentity or homology of amino acid residues of two polypeptides, andadditionally, or alternatively, with respect to the foregoing, theteachings in these references can be used for determining percenthomology: Smith et al., (1981) Adv. Appl. Math. 2:482-489; Smith et al.,(1983) Nucl. Acids Res. 11:2205-2220; Devereux et al., (1984) Nucl.Acids Res. 12:387-395; Feng et al., (1987) J. Molec. Evol. 25:351-360;Higgins et al., (1989) CABIOS 5:151-153; and Thompson et al., (1994)Nucl. Acids Res. 22:4673-4680.

“Having at least X % homolgy”—with respect to two amino acid ornucleotide sequences, refers to the percentage of residues that areidentical in the two sequences when the sequences are optimally aligned.Thus, 90% amino acid sequence identity means that 90% of the amino acidsin two or more optimally aligned polypeptide sequences are identical.

An additional embodiment of the present invention concerns apharmaceutical composition comprising an RTP801 inhibitor in atherapeutically affective amount as an active ingredient and apharmaceutically acceptable carrier. The inhibitor may be a biologicalinhibitor, an organic molecule, a chemical molecule, etc. saidpharmaceutical composition may comprise an RTP801 inhibitor which is apolynucleotide which comprises consecutive nucleotides having a sequencewhich is an antisense sequence to the sequence set forth in FIG. 1 (SEQID No: 1). Further, the RTP801 inhibitor may be a vector comprisingthese polynucleotides. Additionally, the RTP801 inhibitor may be amonoclonal antibody which specifically binds to an epitope comprising4-25 amino acids set forth in FIG. 2 (SEQ ID No:2), or an RNA moleculewhich targets the RTP801 gene mRNA such as an siRNA molecule (optionallydepicted in Tables A-C and in particular, siRNA Nos: 22, 23, 25, 27, 39,41, 42, 49 and 50 of Table A) or a ribozyme.

The active ingredients of the pharmaceutical composition can includeoligonucleotides that are nuclease resistant needed for the practice ofthe invention or a fragment thereof shown to have the same effecttargeted against the appropriate sequence(s) and/or ribozymes.Combinations of active ingredients as disclosed in the present inventioncan be used, including combinations of antisense sequences.

An additional embodiment of the present invention provides for the useof a therapeutically effective dose of an RTP801 inhibitor for thepreparation of a medicament for promoting recovery in a patientsuffering from spinal cord disease or injury. In one embodiment theinhibitor is preferably an siRNA. In another embodiment the inhibitor ispreferably Structure A depicted herein.

The invention has been described in an illustrative manner, and it is tobe understood that the terminology which has been used is intended to bein the nature of words of description rather than of limitation.

Obviously, many modifications and variations of the present inventionare possible in light of the above teachings. It is, therefore, to beunderstood that within the scope of the appended claims, the inventioncan be practiced otherwise than as specifically described.

Throughout this application, various publications, including UnitedStates patents, are referenced by author and year and patents by number.The disclosures of these publications and patents and patentapplications in their entireties are hereby incorporated by referenceinto this application in order to more fully describe the state of theart to which this invention pertains.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 details the coding sequence of the RTP801 gene (SEQ ID NO:1);

FIG. 2 details the amino acid sequence of the RTP801 polypeptide (SEQ IDNO:2);

FIG. 3 is a diagram depicting the exons, CDS, human SNPs and theposition of the various nucleic acid molecules which are either humanspecific or specific for human, mouse and rat in parallel;

FIG. 4A-H depict a panel of Western Blot analysis results obtained uponapplying various double-stranded nucleic acids according to the presentinvention to a first human cell line, whereby the experiment was carriedout twice, referred to as experiment 1 and experiment 2, and whereby theexpression level of p110a and p85 is represented as loading controls andthe intensity (density) of the RTP801 band is a measure for theinhibitory activity of the particular double-stranded nucleic acidapplied;

FIG. 5A-F depict a panel of Western Blot analysis results obtained uponapplying various double-stranded nucleic acids according to the presentinvention to a second human cell line, whereby the experiment wascarried out twice, referred to as experiment 1 and experiment 2, andwhereby the expression level of p110a and p85 is represented as loadingcontrols and the density of the RTP801 band is a measure for theinhibitory activity of the particular double-stranded nucleic acidapplied;

FIG. 6A-C depict a panel of Western Blot analysis results obtained uponapplying various double-stranded nucleic acids according to the presentinvention to the first human cell line at different concentrations,namely 10 nM (5A), 5 nM (5B) and 1 nM (5C), whereby the experiment wascarried out twice, referred to as experiment 1 and experiment 2, andwhereby the expression level of p110a and p85 is represented as loadingcontrols and the density of the RTP801 band is a measure for theinhibitory activity of the particular double-stranded nucleic acidapplied;

FIG. 7 depicts a panel of Western Blot analysis results obtainedapplying various double-stranded nucleic acids according to the presentinvention to a mouse cell line, whereby the experiment was carried outtwice, referred to as experiment 1 and experiment 2, and whereby theexpression level of p110a and p85 is represented as loading controls andthe density of the RTP801 band is a measure for the inhibitory activityof the particular double-stranded nucleic acid applied;

FIG. 8 shows the results of experiments in a mouse AMD model system;

FIG. 9 shows the results of additional experiments in a mouse AMD modelsystem;

FIG. 10 shows the results of experiments in a non-human primate AMDmodel system;

FIG. 11A-B shows the results of additional experiments in a non-humanprimate AMD model system;

FIG. 12A-B shows the results of further additional experiments in anon-human primate AMD model system;

FIG. 13A-B represents an analysis of the experimental results achievedin a non-human primate AMD model;

FIG. 14 represents an additional analysis of the experimental resultsachieved in a non-human primate AMD model.

FIG. 15 A-C shows the results of an experiment involving theintratracheal instillation of an RTP801 expressing plasmid into mice;

FIG. 16 A-C shows the results of a short-term (7 days) cigarette smokingmodel in RTP801 KO and WT mice;

FIG. 17 A-C shows the results of a short-term cigarette smoking model inWT mice instilled with active anti-RTP801 (REDD14) and control (REDD8)siRNA.

FIG. 18 shows the results of experiments with RTP801 KO mice in along-term CS model;

FIG. 19 shows the results of experiments in a mouse ARF model system;

FIG. 20 shows the results of experiments in a mouse Diabetic Retinopathymodel system;

FIG. 21 shows the results of additional experiments in a mouse DiabeticRetinopathy model system;

FIG. 22 shows the results of further additional experiments in a mouseDiabetic Retinopathy model system;

FIG. 23 shows the results of combined RTP801/VEGF inhibition experimentsin a mouse CNV model system;

FIG. 24 shows the results of additional combined RTP801/VEGF inhibitionexperiments in a mouse CNV model system;

FIG. 25 shows the results of experiments studying effect of RTP801 siRNAon gene expression in RPE and neural retina;

FIG. 26 A-B shows additional results of experiments studying effect ofRTP801 siRNA on gene expression in RPE and neural retina; and

FIG. 27 shows the results of experiments demonstrating that RT801NP isas active as RTP801.

EXAMPLES

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, utilize the present invention toits fullest extent. The following preferred specific embodiments are,therefore, to be construed as merely illustrative, and not limitative ofthe claimed invention in any way.

Standard molecular biology protocols known in the art not specificallydescribed herein are generally followed essentially as in Sambrook etal., Molecular cloning: A laboratory manual, Cold Springs HarborLaboratory, New-York (1989, 1992), and in Ausubel et al., CurrentProtocols in Molecular Biology, John Wiley and Sons, Baltimore, Md.(1988).

Standard organic synthesis protocols known in the art not specificallydescribed herein are generally followed essentially as in Organicsyntheses: Vol. 1-79, editors vary, J. Wiley, New York, (1941-2003);Gewert et al., Organic synthesis workbook, Wiley-VCH, Weinheim (2000);Smith & March, Advanced Organic Chemistry, Wiley-Interscience; 5thedition (2001).

Standard medicinal chemistry methods known in the art not specificallydescribed herein are generally followed essentially as in the series“Comprehensive Medicinal Chemistry”, by various authors and editors,published by Pergamon Press.

The features of the present invention disclosed in the specification,the claims and/or the drawings may both separately and in anycombination thereof be material for realizing the invention in variousforms thereof.

Example 1

General Materials and Methods

If not indicated to the contrary, the following materials and methodswere used in Examples 1-5:

Cell Culture

The first human cell line, namely HeLa cells (American Type CultureCollection) were cultured as follows: Hela cells (American Type CultureCollection) were cultured as described in Czaudema F et al. (Czaudema,F., Fechtner, M., Aygun, H., Arnold, W., Klippel, A., Giese, K. &Kaufmann, J. (2003). Nucleic Acids Res, 31, 670-82).

The second human cell line was a human keratinozyte cell line which wascultivated as follows: Human keratinocytes were cultured at 37° C. inDulbecco's modified Eagle medium (DMEM) containing 10% FCS.

The mouse cell line was B16V (American Type Culture Collection) culturedat 37° C. in Dulbecco's modified Eagle medium (DMEM) containing 10% FCS.Culture conditions were as described in Methods Find Exp Clin Pharmacol.1997 May; 19(4):231-9:

In each case, the cells were subject to the experiments as describedherein at a density of about 50,000 cells per well and thedouble-stranded nucleic acid according to the present invention wasadded at 20 nM, whereby the double-stranded nucleic acid was complexedusing 1 μg/ml of a proprietary lipid.

Induction of Hypoxia-Like Condition

The cells were treated with CoCl₂ for inducing a hypoxia-like conditionas follows: siRNA transfections were carried out in 10-cm plates (30-50%confluency) as described by (Czaudema et al., 2003; Kretschmer et al.,2003). Briefly, siRNA were transfected by adding a preformed 10×concentrated complex of GB and lipid in serum-free medium to cells incomplete medium. The total transfection volume was 10 ml. The finallipid concentration was 1.0 μg/ml; the final siRNA concentration was 20nM unless otherwise stated. Induction of the hypoxic responses wascarried out by adding CoCl₂ (100 μM) directly to the tissue culturemedium 24 h before lysis.

Preparation of Cell Extracts and Immuno Blotting

The preparation of cell extracts and immuno blot analysis were carriedout essentially as described by Klippel et al. (Klippel, A., Escobedo,M. A., Wachowicz, M. S., Apell, G., Brown, T. W., Giedlin, M. A.,Kavanaugh, W. M. & Williams, L. T. (1998). Mol Cell Biol, 18, 5699-711;Klippel, A., Reinhard, C., Kavanaugh, W. M., Apell, G., Escobedo, M. A.& Williams, L. T. (1996). Mol Cell Biol, 16, 4117-27). Polyclonalantibodies against full length RTP801 were generated by immunisingrabbits with recombinant RTP801 protein producing bacteria from pET19-bexpression vector (Merck Biosciences GmbH, Schwalbach, Germany). Themurine monoclonal anti-p110a and anti-p85 antibodies have been describedby Klippel et al. (supra).

Example 2

Reduction of RTP801 Expression in a First Human Cell Line

Various double-stranded nucleic acids were prepared. Their locationrelative to the mRNA and CDS as well as human SNPs in the nucleic acidcoding for human RTP801 (databank accession no. NM_(—)019058) isdepicted in FIG. 3. The first human cell line was contacted with saiddouble-stranded nucleic acids as described in example 1. Upon inductionof a hypoxia-like condition and treatment with said double-strandednucleic acids the cells were lysed and the cell lysates subjected toimmunoblotting. p110a, which is a catalytic unit of the P13-kinase, andp85 were used as loading controls. The intensity of the RTP801 band asvisualised using the RTP801 polyclonal antibodies is a measure of theactivity of the individual double-stranded nucleic acids in terms ofreducing the expression level of RTP801.

Each and any of the double-stranded nucleic acids has been such modifiedsuch that a 2′ O-Me group was present on the first, third, fifth,seventh, ninth, eleventh, thirteenth, fifteenth, seventeenth andnineteenth nucleotide of the antisense strand, whereby the very samemodification, i.e. a 2′-O-Me group was present at the second, fourth,sixth, eighth, tenth, twelfth, fourteenth, sixteenth and eighteenthnucleotide of the sense strand. Additionally, it is to be noted that incase of these particular nucleic acids according to the presentinvention the first stretch is identical to the first strand and thesecond stretch is identical to the second strand and these nucleic acidsare also blunt ended.

The experiments were performed twice and the individual results shown inFIGS. 4A to H, where they are designated as experiment 1 and experiment2, respectively.

The representations h, hr and hmr in FIGS. 4A to H indicate that therespective double-stranded nucleic acid was designed such as to addressa section of the RTP801 mRNA which is specific for human RTP801 mRNA(h), to address a section of the RTP801 mRNA which is specific for humanand rat RTP801 mRNA (hr) and to address a section of the RTP801 mRNAwhich is specific for human, mouse and rat RTP801 mRNA (hmr). Thedouble-stranded nucleic acid referred to as no. 40.1 was used as apositive control and untreated cells (UT+) were used as negativecontrol.

In accordance with the results, the following double-stranded nucleicacids turned out to be particularly useful in down-regulating theexpression of RTP801: no. 14, no. 15, no. 20, no. 21, no. 22, no. 23,no. 24, no. 25, no. 27, no. 39, no. 40, no. 41, no. 42, no. 43, no. 44,no. 49 and no. 50 (see Table A).

Example 3

Reduction of RTP801 Expression in a Second Human Cell Line

The experiments as described in connection with Example 2 were repeatedusing the second human cell line as specified in Example 1 and theresults are depicted in FIGS. 5A to F.

As may be deduced from these figures, the results as obtained inconnection with the experiments described in Example 2, were confirmedusing this second human cell line.

Example 4

Dosage Effect of RTP801-Specific Double-Stranded Nucleic Acids

In this experiment, the dosage effect of RTP801-specific double-strandednucleic acids was investigated.

For that purpose, the HeLa cells treated as in connection with Examples2 and 3, whereby the concentration of double-stranded nucleic acid inthe cultivation broth was 10 nM, 5 nM and 1 nM. As positive control,double-stranded nucleic acid no. 40.1 was used, as negative controluntreated cells (UT+). The read out was the same as described inconnection with Examples 2 and 3. The particular double-stranded nucleicacids used were those with internal reference numbers 14, 22, 23 and 27which are directed to stretches on the RTP801 mRNA which are shared byhumans, mice and rats, and double-stranded nucleic acid with internalreference numbers 39 and 42 which are directed to stretches of theRTP801 mRNA specific for human RTP801.

The results are shown in FIG. 6A to C. From said figures it can be takenthat there is a clear concentration dependency of the effect of thedouble-stranded nucleic acids specific for RTP801, whereby the nucleicacid molecules having internal reference numbers 1, 15, 20, 21, 24, 40,41, 43, 44, 22, 23, 27, 39, 42, 40.1, 44.1, and 14, preferably 22, 23,27, 39, 42, 40.1 and 44.1 and more preferably 14, 23 and 27 andpreferably each of said nucleic acid molecule having the particularmodification pattern as described for them in the example part hereinare particularly effective

Example 5

Species Specificity of the RTP801-Specific Double-Stranded Nucleic Acid

The double-stranded nucleic acids according to the present inventionhave been designed against stretches of the RTP801 mRNA which are thesame or different in various species. To test whether there is a speciesspecificity of a RTP801-specific double-stranded nucleic acid, thedouble-stranded nucleic acids with internal reference numbers 14, 22, 23and 27 which address a stretch of the RTP801 mRNA which is conservedamong human, mouse and rat RTP801 mRNA, and the double-stranded nucleicacids with internal reference numbers 39 and 42 which address a stretchof the RTP801 mRNA which is specific for human RTP801 mRNA, i.e. whichaddresses a stretch which as such is not present in mouse or rat, werecompared in terms of down-regulating RTP801 using the same approach andread-out as specified in Examples 1 and 2.

Although all of the double-stranded nucleic acids used are in principleactive against human mRNA and, as shown in the preceding examples, arealso suitable to down-regulate the expression of RTP801, upon using amouse cell line only those double-stranded nucleic acids which are alsospecific for mouse RTP801 mRNA effectively reduced RTP801 expression,namely double-stranded nucleic acids nos. 14, 22, 23 and 27.

From this result it can be concluded that it is possible to designRTP801 addressing double-stranded nucleic acids which are specific forone or several species. This allows use of the very same molecule inanimal models as well as in man.

Example 6

Experimental Models, Methods and Results Relating to MacularDegeneration

The compounds of the present invention were tested in the following ananimal model of Choroidal neovascularization (CNV). This hallmark of wetAMD is induced in model animals by laser treatment.

A) Mouse Model

Choroidal Neovascularization (CNV) Induction

Choroid neovascularization (CNV), a hallmark of wet AMD, was triggeredby laser photocoagulation (532 nm, 200 mW, 100 ms, 75 μm) (OcuLight GL,Iridex, Mountain View, Calif.) performed on both eyes of each mouse onday 0 by a single individual masked to drug group assignment. Laserspots were applied in a standardized fashion around the optic nerve,using a slit lamp delivery system and a cover slip as a contact lens.

Treatment Groups

CNV was induced in the following groups of mice (males 6-8 weeks ofage):

-   -   (1) 12 WT mice;    -   (2) 12 RTP801 Knock-Out mice;    -   (3) 12 WT mice injected with 0.25 μg of synthetic stabilized        active anti-RTP801 siRNA (REDD14) in one eye and inactive        anti-RTP801 siRNA (REDD8—negative control) in the fellow eye, at        days 0 and 7;    -   (4) 12 WT mice injected with 0.25 μg of synthetic stabilized        active anti-RTP801 siRNA (REDD14) in one eye and inactive        anti-GFP siRNA (negative control) in the fellow eye at days 0        and 7;    -   (5) 12 WT mice injected with either 0.1 μg of synthetic        stabilized active anti-RTP801 siRNA (REDD14) in one eye and PBS        (negative control) in the fellow eye at days 0 and 7;    -   (6) 12 WT mice injected with either 0.05 μg of synthetic        stabilized active anti-RTP801 siRNA (REDD14) in one eye and PBS        (negative control) in the fellow eye at days 0 and 7.

Both eyes of each mouse were laser-treated. The volume injected was 2μl.

Evaluation

-   -   1. The experiment was terminated at day 14. For evaluation, the        eyes were enucleated and fixed with 4% paraformaldehyde for 30        min at 4° C. The neurosensory retina was detached and severed        from the optic nerve. The remaining RPE-choroid-sclera complex        was flat mounted in Immu-Mount (Vectashield Mounting Medium,        Vector) and coverslipped. Flat mounts were examined with a        scanning laser confocal microscope (TCS SP, Leica, Germany).        Vessels were visualized by exciting with blue argon laser.        Horizontal optical sections (1 μm step) were obtained from the        surface of the RPE-choroid-sclera complex. The deepest focal        plane in which the surrounding choroidal vascular network        connecting to the lesion could be identified was judged to be        the floor of the lesion. Any vessel in the laser treated area        and superficial to this reference plane was judged as CNV.        Images of each section were digitally stored. The area of        CNV-related fluorescence was measured by computerized image        analysis using the Leica TCS SP software. The summation of whole        fluorescent area in each horizontal section was used as an index        for the volume of CNV.    -   2. Separate WT mice (5 eyes per group) were used for evaluating        RTP801 mRNA expression in CNV (as well as the expression of        other genes relevant to AMD) (untreated and treated with siRNA)        using real-time PCR on RNA extracted from RPE/choroids, or from        neural retina.        Results    -   1. RTP801 KO mice displayed 30% less blood vessel leakage        compared to WT mice following CNV induction; see FIG. 8.    -   2. Synthetic stabilized siRNA against RTP801, REDD14, elicited a        dose-dependent reduction of the CNV volume. A maximum of ˜70%        inhibition compared to PBS-injected eyes was achieved at a        REDD14 (sequence No. 14 in table 1, SEQ ID No.s 16 (sense) and        66 (antisense)) dose of 0.25 ug per eye. At the same dose, both        negative control siRNAs, REDD8 and anti-GFP siRNA, displayed        only 27% and 33% CNV volume reduction respectively, supporting        both the superior efficacy of REDD14 and also the specificity of        its effect.        B) Non-Human Primate Model        CNV Induction

Eight male cynomoglus monkeys (Macaca fascicularis) 2-6 years of agewere used for the study. Choroidal neovascularization (CNV) was inducedby perimacular laser treatment of both eyes prior to doseadministration. Nine lesions were placed in the macula with a laser[OcuLight GL (532 nm) Laser Photo-coagulator with an IRIS Medical®Portable Slit Lamp Adaptor], and laser spots in the right eye weremirror the placement in the left eye. The approximate laser parameterswere as follows: spot size: 50-100 μm diameter; laser power: 300-700milliwatts; exposure time: 0.1 seconds.

Treatment

Immediately following laser treatment, both eyes of all animals weresubjected to a single intravitreal injection. Left eye was dosed with350 ug of synthetic stabilized siRNA against RTP801 (the same one usedin the mouse study) in the final volume of 50 ul, whereas thecontralateral eye received 50 ul of PBS (vehicle).

Evaluation

-   -   1. All the animals were subjected to daily examination of food        consumption and body weight measurements.    -   2. 2 monkeys were euthanized at day 6 following CNV induction.        Their eyes were enucleated and posterior pole was flattened.        Then the fovea region was excised and separated into choroids        and neuroretina which were separately (for every animal) frozen        in liquid nitrogen to be subsequently used for RNA extraction        and real time PCR evaluation of RTP801 expression.    -   3. Fluorescein angiograms were performed pre-study, and at the        end of weeks 1, 2, and 3 following CNV induction. Photographs        were taken, using a fundus camera (TRC-50EX Retina Camera).        Images were captured using the TOPCON IMAGEnet™ system.        Fluorescein dye (10% fluorescein sodium, approximately 0.1        mL/kg) was injected via vascular access ports. Photographs were        taken at several timepoints following dye injection, to include        the arterial phase, early arteriovenous phase and several late        arteriovenous phases in order to evaluate neovascularization snd        to monitor leakage of fluorescein associated with CNV lesions.        Interpretation and analysis of the fluorescein angiograms was        independently conducted by two ophthalmologists.        -   Neovascularization (NV) was assessed in early angiograms and            every spot was graded according to the following scheme:        -   0—no signs of NV        -   0.5—suspicious spot        -   1′—“hot” spot        -   2—NV in the laser burn        -   3—evident NV        -   Leakage was assessed according to the following scheme:        -   0—no leakage        -   0.5—suspicious spot        -   1—evident small spot leakage        -   2—leakage growing with time        -   3—leakage greater than previous borders (evidently)

In addition, the size of every spot was compared between the early andthe late angiograms using morphometric measurements, and the increase inthe spot's size resulting from the leakage was calculated.

-   -   4. Electroretinograms (ERGs) were recorded using an Epic 2000        electroretinograph according to Sierra's SOPs and the        study-specific SOP, including the use of the Ganzfield        apparatus, at prestudy and in the end of week 3 The tabulated        ERG data were evaluated by a veterinary ophthalmologist.

The study was terminated at day 21 post CNV induction. Gross necropsyand histological examination were performed on organs and tissuesincluding the eyes.

Results

-   1. siRNA against RTP801 reduced RTP801 expression in the    RPE/choroids of laser-treated animals, as measured at day 6 post CNV    induction by real-time PCR (see FIG. 10).-   2. Comparison of the spot grading for leakage and neovascularization    between the fellow eyes in each individual monkey revealed that both    of these pathological characteristics were diminished in the eyes    injected with RTP801 siRNA as compared to the control (for leakage    results, see FIG. 11; for neovascularization results, see FIG. 12).-   3. Calculation of the overall number of spots with higher    clinically-relevant grades (2 and 3) of leakage or    neovascularization in all siRNA-injected eyes compared to all    PBS-injected eyes again revealed that siRNA injected eyes were less    affected (see FIG. 13, a+b).-   4. The overall grading data for leakage of spots and    neovascularisation was subjected to statistical evaluation. The    existence of differences between the siRNA and control treatments    was analyzed by calculating the delta between the mean spot ranks of    the control right (R) eye and siRNA-injected left (L) eye    (delta=R−L). The significance of the difference was calculated using    a non-parametric statistical method, Wilcoxon signed ranks test—a    one tail test. Different phases of angiograms (early arterial,    arterio-venous and late venous) were analyzed separately for every    week (1, 2, and 3).

Table 1 shows the significance (one tail test) of leakage rankdifference from 0 for each group (p-values <0.05 are underlined). Asignificant leakage rank reduction was found in the left eyes (siRNAtreated) with respect to the right (Placebo treated) in week 2 and 3 inthe late angiograms. TABLE 1 P - Value Wilcoxon Leakage Signed RankAngiograms Week Test Early 1 0.2500 2 0.5000 3 0.5000 Arterio-Venus 10.3438 2 0.1250 3 0.2344 Late 1 0.1250 2 0.0313 3 0.0156

-   -   Note that late angiograms are usually utilized for evaluation of        leakage parameters.

Table 2 shows the significance (one tail test) of neovascularization(NV) rank difference from 0 for each group (p-values <0.05 areunderlined). TABLE 2 P - Value Wilcoxon NV Signed Rank Angiograms WeekTest Early 1 0.0781 2 0.0313 3 0.0313 Arterio Venus 1 0.0625 2 0.0313 30.1563 Late 1 0.2500 2 0.3438 3 0.2500

A significant NV rank reduction was found in the left eyes with respectto the right in week 2 and 3 in the early period and in the ArterioVenus period in week 2.

Note that early angiograms are usually utilized for evaluation ofneovascularization parameters.

-   5. Quantitative morphometric evaluation of the increase in area of    the spots occurring between early (arterial phase) and late (venous    phase) angiograms due to the leakage revealed that this parameter    was significantly reduced in the laser spots within siRNA-injected    eyes (left eyes, OS) compared to control (right eyes, OD). Two    examples are shown in FIG. 14. The graphs demonstrate the relative    increase (in %) in the area of every spot in the left and right eye    of animals #3315 and 3300.

Additionally, it was noted throughout all the above studies that antiRTP-801 siRNA had no adverse effects on electroretinograms (ERG), on eyehistology or on structure and function of other organs and systems.

To Summarize the Above Experiments and Results:

-   -   1. Both genetic (RTP801−/−) and therapeutic siRNA inhibition of        RTP801 expression in the laser-induced CNV model of wet        age-related macular degeneration (wet AMD) result in significant        reduction of the CNV volume.    -   2. Positive results were obtained in mouse and non-human primate        model.    -   3. Pathological and ERG examination in monkey did not reveal any        siRNA-mediated toxicity either in eyes or in any other organs or        systems.        C) Efficacy of Combination Therapy of RTP801 siRNA (REDD14) and        Anti-VEGF Antibody

The efficacy of combination therapy of RTP801 siRNA (REDD14) andanti-VEGF antibody in the treatment of diseases in which CNV occurs wastested in the above mouse CNV model.

A) CNV Volume Studies

The volume of choroidal neovascularization (CNV) 3 weeks after laserinjury was computed by confocal fluorescence microscopy as previouslydescribed (Sakurai et al. IOVS 2003;44: 3578-85 & Sakurai et al. IOVS2003; 44: 2743-2749).

In previous studies we found that anti-VEGF-A antibody (Ab) reduced CNVvolume in a dose dependent fashion. A dose of 1 ng of VEGF-A Ab waschosen for the REDD14+VEGF-A Ab combination studies because this dosehad an intermediate inhibitory effect: VEGF-A Ab (1 ng) reduced the sizeof CNV by 26±6%.

The principal findings of the REDD14+VEGF-A antibody (Ab) study are:

-   -   The addition of REDD14 at the lower 0.05 μg dose reduced the        size of CNV by 27±4% compared to VEGF-A Ab alone.    -   The addition of REDD14 at the higher 0.25 μg dose reduced the        size of CNV by 55±3% compared to VEGF-A Ab alone.        B) CNV Leakage Studies        Experiment 1

This experiment was designed in order to identify a potential additiveor synergistic therapeutic effect of inhibition of VEGF and RTP801 inthe model of laser-induced choroid neovascularization in mice

Materials:

-   -   REDD14 (RTP801 siRNA)    -   REDD8 (negative control)    -   Anti-VEGF antibodies    -   Non-specific IgG (negative control)

CNV was induced on day zero as described above; the test material wasinjected to the subjects on day zero and day 7.

The results were evaluated by Fluorescein angiography on weeks 1, 2, 3,and by CNV volume measurement on week 3. each test group was composed of10 eyes.

Experimental Groups:

-   -   VEGF Ab 0.5 ng/eye    -   VEGF Ab 1 ng/eye    -   VEGF Ab 2 ng/eye    -   VEGF Ab 4 ng/eye    -   REDD14 0.05 ug/eye    -   REDD14 0.1 ug/eye    -   REDD14 0.25 ug/eye    -   REDD14 0.05 ug/eye+VEGF Ab 1 ng/eye    -   REDD14 0.1 ug/eye+VEGF Ab 1 ng/eye    -   REDD14 0.25 ug/eye+VEGF Ab 1 ng/eye        Control Groups    -   PBS    -   Non-specific IgG 2 ng/eye    -   REDD8 0.1 ug/eye    -   REDD8 0.1 ug/eye+VEGF Ab 1 ng/eye        Results

The results of the above experiment are presented in FIGS. 23-24. Theseresults show that simultaneous intravitreal administration of VEGF Aband REDD14 leads to augmented and dose-dependent inhibition of Choroidneovascularization and Choroid blood vessel leakage, as expressed inreduced incidence of Grade 4 spots and increased incidence of Grade 1spots. Angiograms were graded using a modification of asemi-quantitative grading (1-4) scheme previously published (Sakurai etal. IOVS 2003; 44: 2743-2749). Grade 1 lesions are considered as neverhaving formed, i.e., equivalent to complete prevention. Grade 4 lesionsare considered pathologically significant, i.e., equivalent to lesionsthat would be treated in patients. VEGF-A Ab (1 ng) reduced theincidence of Grade 4 lesions per eye by 38±8% and increased theincidence of Grade 1 lesions per eye by 66±43%.

The principal findings of the REDD14+VEGF-A Ab combination leakage studyare:

-   -   The addition of REDD14 at the lower 0.05 μg dose reduced the        incidence of Grade 4 lesions by 66±12% compared to VEGF-A Ab        alone.    -   The addition of REDD14 at the higher 0.25 μg dose reduced the        incidence of Grade 4 lesions by 60±12% compared to VEGF-A Ab        alone.    -   The addition of REDD14 at the higher 0.25 μg dose doubled        (100±34%) the incidence of Grade 1 lesions compared to VEGF-A Ab        alone.        Experiment 2

This experiment was designed in order to study the effect of REDD14 ongene expression in RPE and neural retina.

Experimental Design

Groups:

-   -   PBS    -   REDD14 0.25 mg

The Group size was 5 eyes. CNV was induced by laser treatment asdescribed above on day zero; the test material was also injected on dayzero, and the effect evaluated by qPCR analysis of gene expression inRPE and neural retina on days zero and 5.

Results

The results of the above experiment are presented in FIG. 25. Theseresults show that the administration of REDD14 causes:

-   -   ˜40% downregulation of RTP801 expression below the baseline both        in RPE and in neural retina (see also FIG. 26);    -   ˜70% upregulation of PEDF expression over the baseline in neural        retina (note: in PBS-injected eyes expression of PEDF is 40%        downregulated below the baseline)    -   ˜40% downregulation of VEGF 164 expression below the baseline in        RPE (note: in PBS-injected eyes, expression of VEGF164 is 20%        down-regulated)    -   ˜50% reduction of MCP1 expression in RPE/choroids (FIG. 26)        General Conclusions from Both Experiments:    -   Simultaneous inhibition of RTP801 and VEGF has enhanced        inhibitory effect on choroid neovascularization and neovascular        leakage.    -   Inhibition of RTP801 expression by REDD14 not only prevents PEDF        downregulation in the CNV model but enhances its expression        compared to the baseline.    -   Inhibition of RTP801 expression leads to concomitant        downregulation of MCP1 which should have an anti-inflammatory        effect.    -   Without being bound by theory, the increase of PEDF expression        by REDD14 may underlie the observed cooperative effect of        simultaneous inhibition of VEGF and RTP801        -   (Note: PEDF is a well-known antiangiogenic and            neuroprotective factor.)    -   Without being bound by theory, the reduction of MCP1 expression        by REDD14 may also underlie the observed cooperative effect of        simultaneous inhibition of VEGF and RTP801        -   (Note: MCP1 is a known pro-inflammatory chemokine involved            in pathogenesis of AMD.)

Additional AMD models which may be used to test the methods of thepresent invention:

-   -   Ccl-2 or Ccr-2 deficient animals—deficiency in either of these        proteins causes the development of some of the main features of        AMD. Animals deficient in these proteins can be used to test the        methods of the present invention.

For further information on AMD animal models, see: Chader, Visionresearch 42 (2002) 393-399; Ambati et al., Nature Medicine 9(11) (2003)1390-1397; Tolentino et al., Retina 24 (2004) 132-138.

D) Comparison of Activity of REDD14 Anti RTP801 siRNA Possessing a3′Phosphate Group on each Strand with the Same Molecule Lacking 3′Phosphates (REDD14NP) in the Laser-Induced CNV Model.

The experiment was generally performed and evaluated as described above.One eye of each mouse (12 per group) was injected with 0.25 ug of REDD14siRNA whereas another eye was injected with REDD14NP siRNA.

Results

Both siRNAs equally efficiently reduced CNV volume (FIG. 27).

Example 7

Models and Results Relating to COPD and Emphysema

The compounds of the present invention were tested in the following ananimal models:

-   -   Cigarette smoke-induced emphysema model: chronic exposure to        cigarette smoke causes emphysema in several animals such as,        inter alia, mouse, guinea pig.    -   Lung protease activity as a trigger of emphysema.    -   VEGFR inhibition model of emphysema.    -   Bronchial instillation with human neutrophil/pancreatic elastase        in rodents.    -   MMP (matrix metalloprotease)-induced enphysema.    -   Inflammation-induced emphysema.

Additionally, emphysema models may be generated through genetic means(e.g., mice carrying the TSK mutation), and emphysematous animals may begenerated by known modifiers of susceptibility to emphysema such as,inter alia, lung injury, alveolar hypoplasia, hyperoxia, glucocorticoidtreatment and nutrition.

A. Evaluation of the Influence of Lack of RTP801 on Disease Developmentin Mouse Models of Emphysema (Using RTP801 Knockout Mice)

-   (1) Cigarette smoking (CS) induced inflammation and apoptosis is    initiated in 5 RTP801 KO and 5 control wild type 4 months old male    mice. The mice are subjected to intense CS (as described in    Rangasamy et al., see above) for 7 days. KO and WT non-treated mice    from the VEGFR inhibition experiment above can also serve as    non-treated control groups for this experiment. The lungs are    subsequently agarose-inflated, fixed and imbedded in paraffin, and    development oxidative stress in the KO mice is assessed by:    -   a) immunohistochemical localization and quantitation of 8-oxo-dG        in the lung sections;    -   b) immunohistochemical localization and quantitation of active        caspase 3 in the lung sections using specific antibodies, or        quantitative evaluation of the number of TUNEL-positive cells;    -   c) measurement of ceramide concentration in the lung extracts;    -   d) measurement of caspase activity in the lung extracts.-   (2) Long-term cigarette smoking in the KO mice.

6 KO and 6 age-matched WT female mice were subjected to intensecigarette smoking (5 hrs a day) during a period of 6 months. The micewere then sacrificed, and average interseptal diameter (a parameter ofemphysema development) was evaluated using a morphometric approach.

B. Evaluation of the Influence of Lack of RTP801 on Disease Progressionin Mouse Models of Emphysema by Inhibiting Endogenous RTP801 EmployingIntralung Delivery RTP801-Inactivating siRNA

CS-induced inflammation was induced by 7 day smoking in 2 groups ofC57BL6 mice, 10 mice per group. Group 1: CS+delivery of control siRNA(REDD8) siRNA; Group 2: CS+RTP801 siRNA (REDD14). Control groups of micewere instilled with either type of siRNA but kept in room airconditions. The animals were evaluated as in the above experiment withthe Knock-Out mice.

Methods

Exposure to Cigarette Smoking (CS)

Exposure is carried out (7 h/day, 7 days/week) by burning 2R4F referencecigarettes (2.45 mg nicotine per cigarette; purchased from the TobaccoResearch Institute, University of Kentucky, Lexington, Ky., USA) using asmoking machine (Model TE-10, Teague Enterprises, Davis, Calif., USA).Each smoldering cigarette was puffed for 2 s, once every minute for atotal of eight puffs, at a flow rate of 1.05 L/min, to provide astandard puff of 35 cm3. The smoke machine is adjusted to produce amixture of sidestream smoke (89%) and mainstream smoke (11%) by burningfive cigarettes at one time. Chamber atmosphere is monitored for totalsuspended particulates and carbon monoxide, with concentrations of 90mg/m3 and 350 ppm, respectively.

Morphologic and Morphometric Analyses

After exposing the mice to CS or instillation of RTP801 expressingplasmid, the mice are anesthetized with halothane and the lungs areinflated with 0.5% low-melting agarose at a constant pressure of 25 cmas previously described⁶. The inflated lungs are fixed in 10% bufferedformalin and embedded in paraffin. Sections (5 μm) are stained withhematoxylin and eosin. Mean alveolar diameter, alveolar length, and meanlinear intercepts are determined by computer-assisted morphometry withthe Image Pro Plus software (Media Cybernetics, Silver Spring, Md.,USA). The lung sections in each group are coded and representativeimages (15 per lung section) are acquired by an investigator masked tothe identity of the slides, with a Nikon E800 microscope, 20× lens.

Bronchoalveolar Lavage (BAL) and Phenotyping

Following exposure to CS or instillation of RTP801 expressing plasmid,the mice are anesthetized with sodium pentobarbital. The BAL fluidcollected from the lungs of the mice is centrifuged (500 ′g at 4° C.),and the cell pellet is resuspended in phosphate-buffered saline. Thetotal number of cells in the lavage fluid is determined, and 2×104 cellsare cytocentrifuged (Shandon Southern Products, Pittsburgh, Pa., USA)onto glass slides and stained with Wright-Giemsa stain. Differentialcell counts are performed on 300 cells, according to standard cytologictechniques.

Identification of Alveolar Apoptotic Cell Populations in the Lungs.

To identify the different alveolar cell types undergoing apoptosis inthe lungs, an immunohistochemical staining of active caspase 3 isperformed in the lung sections from the room air (RA) as well as CSexposed mice. To identify the apoptotic type II epithelial cells in thelungs, after active caspase 3 labeling, the lung sections are incubatedfirst with anti-mouse surfactant protein C (SpC) antibody and then withan anti-rabbit Texas red antibody. Apoptotic endothelial cells areidentified by incubating the sections first with the anti-mouse CD 31antibody and then with the biotinylated rabbit anti-mouse secondaryantibody. The lung sections are rinsed in PBS and then incubated withthe streptavidin-Texas red conjugated complex. The apoptotic macrophagesin the lungs are identified by incubating the sections first with therat anti-mouse Mac-3 antibody and then with the anti-rat Texas redantibody. Finally, DAPI is applied to all lung sections, incubated for 5minutes, washed and mounted with Vectashield HardSet mounting medium.DAPI and fluorescein are visualized at 330-380 nm and 465-495 nm,respectively. Images of the lung sections are acquired with the NikonE800 microscope, 40× lens.

Immunohistochemical Localization of Active Caspase-3

Immunohistochemical staining of active caspase-3 assay is performedusing anti-active caspase-3 antibody and the active caspase-3-positivecells are counted with a macro, using Image Pro Plus program. The countsare normalized by the sum of the alveolar profiles herein named asalveolar length and expressed in μm. Alveolar length correlatesinversely with mean linear intercept, i.e., as the alveolar septa aredestroyed, mean linear intercepts increases as total alveolar length,i.e., total alveolar septal length decreases.

Caspase 3 Activity Assay

The caspase-3/7 activity is measured in lung tissue extracts using afluorometric assay according to the manufacturer's instructions.Snap-frozen lung tissue (n=3 per group) was homogenized with the assaybuffer, followed by sonication and centrifugation at 800×g. Afterremoval of nuclei and cellular debris, the supernatant (300 μg protein)is then incubated with the pro-fluorescent substrate at room temperaturefor 1 h and the fluorescence intensity was measured utilizing a Typhoonphosphoimager (Amersham Biosciences, Inc., Piscataway, N.J., USA). Theresults are expressed as the rate of specific caspase-3 substratecleavage, expressed in units of caspase 3 enzymatic activity, normalizedby total protein concentration. Active recombinant caspase 3 wasutilized as the assay standard (0-4 U). Tissue lysates withoutsubstrate, assay buffer alone, and lysates with caspase 3 inhibitor wereutilized as negative controls.

Immunohistochemical Localization of 8-oxo-dG

For the immunohistochemical localization and quantification of 8-oxo-dG,lung sections from the mice exposed to CS or instilled with RTP801expressing plasmid are incubated with anti-8-oxo-dG antibody and stainedusing InnoGenex™ Iso-IHC DAB kit using mouse antibodies. The8-oxo-dG-positive cells are counted with a macro (using Image Pro Plus),and the counts were normalized by alveolar length as described.

Instillation of Plasmid DNA into Mouse Lungs

Plasmid DNA of RTP801 expressing and control vectors were prepared underendotoxin-free DNA isolation kit. For intra-tracheal instillation, 50 ugof plasmid DNA is delivered in 80 ul sterile perfluorocarbon. The oxygencarrying properties of perfluorocarbon make it well-tolerated at thesevolumes, while its physical-chemical properties allow for extremelyefficient distal lung delivery when instilled intratracheally. Mice areanesthetized by brief inhalational halothane exposure, the tongue isgently pulled forward by forceps and the trachea instilled withperfluorocarbon solution applied at the base of the tongue via a bluntangiocatheter.

Instillation of siRNA into Mouse Lungs.

Mice are anesthetized with an intra-peritoneal injection ofKetamine/Xylazine (115/22 mg/kg). 50 μg of siRNA is instilledintranasally in 50 μl volume of 0.9% NaCl by delivering five consecutive10 μl portions. At the end of the intranasal instillation, the mouse'shead is held straight up for 1 minute to ensure that all the instilledsolution drains inside.

For further information, see: Rangasamy T, Cho C Y, Thimmulappa, R K,Zhen L, Srisuma S S, Kensler T W, Yamamoto M, Petrache I, Tuder R M,Biswal S. Genetic ablation of Nrf2 enhances susceptibility to cigarettesmoke-iduced emphysema in mice. Submitted to Journal of ClinincalInvestigation; Yasunori Kasahara, Rubin M. Tuder, Carlyne D. Cool, DavidA. Lynch, Sonia C. Flores, and Norbert F. Voelkel. Endothelial CellDeath and Decreased Expression of Vascular Endothelial Growth Factor andVascular Endothelial Growth Factor Receptor 2 in Emphysema. Am J RespirCrit Care Med Vol 163. pp 737-744, 2001; Yasunori Kasahara, Rubin M.Tuder, Laimute Taraseviciene-Stewart, Timothy D. Le Cras, Steven Abman,Peter K. Hirth, Johannes Waltenberger, and Norbert F. Voelkel.Inhibition of VEGF receptors causes lung cell apoptosis and emphysema.J. Clin. Invest. 106:1311-1319 (2000); and a review on the topic: RobinM. Tuder, Sharon McGrath and Enid Neptune, The pathological mechanismsof emphysema models: what do they have in common?, PulmonaryPharmacology & Therpaeutics 2002.

Results

-   -   1. Instillation of an RTP801 expressing plasmid results in an        emphysema-like phenotype in mouse lungs which is evident by (1)        inctease in bronchoalveolar lavage cell counts (FIG. 15 a); (2)        apoptosis of lung septal cells (FIG. 15 b) and increase in the        alveolar diameter (FIG. 15 c).    -   2. Instillation of RTP801 siRNA (REDD14) results in reduction of        RTP801 expression in the lungs (FIG. 17 b).    -   3. RTP801 KO mice are protected from emphysema development        following 6 months of cigarette smoking as evident by the lack        of enlargement of alveolar diameter. (FIG. 18).    -   4. RTP801 KO mice are protected from cigarette smoking induced        inflammation as evident by reduced number of inflammatory        bronchoalveolage cells following 1 week of cigarette smoking        (FIG. 16, a−b).    -   5. RTP801 KO mice are protected from cigarette smoking induced        apoptosis of lung septal cells as evidenced by lung section        staining for activated caspase (FIG. 16 c).    -   6. REDD14-instilled mice are partially protected from cigarette        smoking induced inflammation as evident by reduced number of        inflammatory bronchoalveolage cells following 1 week of        cigarette smoking (FIG. 17 a).    -   7. REDD14-instilled mice are partially protected from cigarette        smoking induced apoptosis of lung septal cells as evidenced by        lung section staining for activated caspase and by        immunoblotting of lung extracts with anti-activated caspase 3        antibodies ((FIG. 17 c)

Example 8

Models and Results Relating to Microvascular Disorders

The compounds of the present invention were tested in animal models of arange of microvascular disorders as described below.

1. Diabetic Retinopathy

RTP801 promotes neuronal cell apoptosis and generation of reactiveoxygen species in vitro. The inventor of the current invention alsofound that in RTP801 knockout (KO) mice subjected to the model ofretinopathy of prematurity (ROP), pathologic neovascularization NV wasreduced under hypoxic conditions, despite elevations in VEGF, whereasthe lack of this gene did not influence physiologic neonatal retinal NV.Moreover, in this model, lack of RTP801 was also protective againsthypoxic neuronal apoptosis and hyperoxic vaso-obliteration.

Experiment 1

Diabetes was induced in 8 wk old RTP801 KO and C57/129sv wildtype (WT)littermate mice by intraperitoneal injection of STZ. After 4 weeks, ERG(single white flash, 1.4×10ˆ4 ftc, 5 ms) was obtained from the left eyeafter 1 hour of dark adaptation. RVP was assessed from both eyes usingthe Evans-blue albumin permeation technique.

Results

Blood glucose was not different between diabetic (DM) WT and DM KO(495±109 vs 513±76 mg/dl), nor nondiabetic (NDM) WT and KO (130±10 vs135±31 mg/dl, respectively). RVP in the DM WT group was increased 138%(51.2±37.9 μL/g/hr, n=8) compared to NDM WT (21.5±18.8 μL/g/hr, n=9,p=0.055). In contrast, RVP was reduced by 80% in DM KO (9.5±8.5 μL/g/hr,n=6, p=0.023) as compared to the DM WT mice, resulting in a 140%decrease of diabetes-induced RVP. In DM WT mice, there was aprolongation (p<0.05) of the oscillatory potential implicit times forOP2 (11%), OP3 (12%), & OP4 (14%) and for the B-wave (23%) as comparedto NDM WT. A-wave was not significantly changed. These changes werenormalized ˜100% in DM KO mice for OP3 & OP4 and 65% for B-wave ascompared to NDM KO. Conclusion: Knock out of RTP801 amelioratesdiabetes-induced RVP and ERG abnormalities in mice, suggesting that thishypoxia inducible gene may serve an important role in the pathogenesisof early diabetic retinal disease.

Experiment 2

Diabetes was induced in RTP801 knockout and in control wild type micewith the matched genetic background. In addition, it was induced inC57B16 mice, which were subsequently used for intravitreal injection ofanti-RTP801 and control siRNAs. For diabetes induction, the mice wereinjected with streptozotocin (STZ 90 mg/kg/d for 2 days after overnightfast). Animal physiology was monitored throughout the study for changesin blood glucose, body weight, and hematocrit. Vehicle-injected miceserved as controls. The appropriate animals were treated by intravitrealinjections of 1 ug of REDD14 anti-RTP801 siRNA or 1 ug of anti-GFPcontrol siRNA. siRNA was injected twice in the course of the study—onday 0, when the first STZ injection was performed, and on day 14 afterthe STZ injection.

Retinal vascular leakage was measured using the Evans-blue (EB) dyetechnique on the animals after 4 weeks duration of diabetes. Mice had acatheter implanted into the right jugular vein 24 hours prior to EvansBlue (EB) measurements. Retinal permeability measurements in both eyesof each animal followed a standard Evans-blue protocol.

Results

-   1. Retinal blood vessel leakage was reduced by 70% in RTP801 KO    diabetic mice compared with wild type diabetic mice (see FIG. 20).-   2. The Knock out of RTP801 normalizes ERG abnormalities in mice: In    DM WT mice, there was a prolongation (p<0.05) of the oscillatory    potential implicit times for OP2 (11%), OP3 (12%), & OP4 (14%) and    for the B-wave (23%) as compared to NDM WT. A-wave was not    significantly changed. These changes were normalized ˜100% in DM    RTP801 KO mice for OP3 & OP4 and 65% for B-wave as compared to NDM    RTP801 KO (see FIG. 21).-   3. Similarly to the results in KO mice, retinal blood vessel leakage    was reduced by 50% in diabetic mice injected intravitreally with    REDD14 siRNA against RTP801 compared to diabetic mice intraviterally    injected with control siRNA against GFP (see FIG. 22).    2. Retinopathy of Prematurity

Retinopathy of prematurity was induced by exposing the test animals tohypoxic and hyperoxic conditions, and subsequently testing the effectson the retina. Results showed that RTP801 KO mice were protected fromretinopathy of prematurity, thereby validating the protective effect ofRTP801 inhibition.

3. Myocardial Infarction

Myocardial infarction was induced by Left Anterior Descending arteryligation in mice, both short term and long term. Results: reduction ofTnT and CPK-MB fraction levels at 24 hrs postinfarct in the blood andbetter echocardiogram (ejection fraction volume) at 28 days postinfarctin RTP801 KO mice.

4. Microvascular Ischemic Conditions

Animal models for assessing ischemic conditions include:

-   1. Closed Head Injury (CHI)—Experimental TBI produces a series of    events contributing to neurological and neurometabolic cascades,    which are related to the degree and extent of behavioral deficits.    CHI is induced under anesthesia, while a weight is allowed to    free-fall from a prefixed height (Chen et al, J. Neurotrauma 13,    557, 1996) over the exposed skull covering the left hemisphere in    the midcoronal plane.-   2. Transient middle cerebral artery occlusion (MCAO)— a 90 to 120    minutes transient focal ischemia is performed in adult, male Sprague    Dawley rats, 300-370 gr. The method employed is the intraluminal    suture MCAO (Longa et al., Stroke, 30, 84, 1989, and Dogan et    al., J. Neurochem. 72, 765, 1999). Briefly, under halothane    anesthesia, a 3-0-nylon suture material coated with Poly-L-Lysine is    inserted into the right internal carotid artery (ICA) through a hole    in the external carotid artery. The nylon thread is pushed into the    ICA to the right MCA origin (20-23 mm). 90-120 minutes later the    thread is pulled off, the animal is closed and allowed to recover.-   3. Permanent middle cerebral artery occlusion (MCAO)—occlusion is    permanent, unilateral-induced by electrocoagulation of MCA. Both    methods lead to focal brain ischemia of the ipsilateral side of the    brain cortex leaving the contralateral side intact (control). The    left MCA is exposed via a temporal craniectomy, as described for    rats by Tamura A. et al., J Cereb Blood Flow Metab. 1981; 1:53-60.    The MCA and its lenticulostriatal branch are occluded proximally to    the medial border of the olfactory tract with microbipolar    coagulation. The wound is sutured, and animals returned to their    home cage in a room warmed at 26° C. to 28° C. The temperature of    the animals is maintained all the time with an automatic thermostat.    5. Acute Renal Failure (ARF)

Testing active siRNA for treating ARF may be done using sepsis-inducedARF or ischemia-reperfusion-induced ARF.

1. Sepsis Induced ARF

Two predictive animal models of sepsis-induced ARF are described byMiyaji T, Hu X, Yuen P S, Muramatsu Y, Iyer S, Hewitt S M, Star R A,2003, Ethyl pyruvate decreases sepsis-induced acute renal failure andmultiple organ damage in aged mice, Kidney Int. Nov; 64(5):1620-31.These two models are lipopolysaccharide administration and cecalligation puncture in mice, preferably in aged mice.

2. Ischemia-Reperfusion-Induced ARF

This predictive animal model is described by Kelly K J, Plotkin Z,Vulgamott S L, Dagher P C, 2003 January. P53 mediates the apoptoticresponse to GTP depletion after renal ischemia-reperfusion: protectiverole of a p53 inhibitor, J Am Soc Nephrol.; 14(1):128-38.

Ischemia-reperfusion injury was induced in rats following 45 minutesbilateral kidney arterial clamp and subsequent release of the clamp toallow 24 hours of reperfusion. 250 μg of REDD14 or GFP siRNA (negativecontrol) were injected into the jugular vein 2 hrs prior to and 30minutes following the clamp. Additional 250 μg of siRNA were given viathe tail vein at 4 and 8 hrs after the clamp. siRNA against GFP servedas a negative control. ARF progression was monitored by measurement ofserum creatinine levels before and 24 hrs post surgery. At the end ofthe experiment, the rats were perfused via an indwelling femoral linewith warm PBS followed by 4% paraformaldehyde. The left kidneys wereremoved and stored in 4% paraformaldehyde for subsequent histologicalanalysis. Acute renal failure is frequently defined as an acute increaseof the serum creatinine level from baseline. An increase of at least 0.5mg per dL or 44.2 μmol per L of serum creatinine is considered as anindication for acute renal failure. Serum creatinine was measured attime zero before the surgery and at 24 hours post ARF surgery.

To study the distribution of siRNA in the rat kidney, Cy3-labeled 19-merblunt-ended siRNA molecules (2 mg/kg) having alternating O-methylmodification in the sugar residues were administered iv for 3-5 min,after which in vivo imaging was conducted using two-photon confocalmicroscopy. The confocal microscopy analysis revealed that the majorityof siRNA in the kidneys is concentrated in the endosomal compartment ofproximal tubular cells. Both endosomal and cytoplasmic siRNAfluorescence were relatively stable during the first 2 hrs post deliveryand disappeared at 24 hrs.

As evident from FIG. 19, there was a ten-fold increase in the level ofserum creatinine following a 45-min of kidney bilateral arterial clamptreatment (PBS treatment). Four injections of 801 siRNA (REDD14, SEQ InNo.s 16 and 66) (2 hrs prior to the clamp and 30 min, 4 h and 8 h afterthe clamp) significantly reduced the creatinine level in serum by 40%(P<0.02). These results suggest that 801 siRNA can protect renal tissuefrom the effects of ischemia-reperfusion injury and thus reduce theseverity of ARF.

Example 9

Preparation of siRNAs

Using proprietary algorithms and the known sequence of gene RTP801 (SEQID NO:1), the sequences of many potential siRNAs were generated. siRNAmolecules according to the above specifications were preparedessentially as described herein.

The siRNAs of the present invention can be synthesized by any of themethods which are well-known in the art for synthesis of ribonucleic (ordeoxyribonucleic) oligonucleotides. For example, a commerciallyavailable machine (available, inter alia, from Applied Biosystems) canbe used; the oligonucleotides are prepared according to the sequencesdisclosed herein. Overlapping pairs of chemically synthesized fragmentscan be ligated using methods well known in the art (e.g., see U.S. Pat.No. 6,121,426). The strands are synthesized separately and then areannealed to each other in the tube. Then, the double-stranded siRNAs areseparated from the single-stranded oligonucleotides that were notannealed (e.g. because of the excess of one of them) by HPLC. Inrelation to the siRNAs or siRNA fragments of the present invention, twoor more such sequences can be synthesized and linked together for use inthe present invention.

The siRNA molecules of the invention may be synthesized by proceduresknown in the art e.g. the procedures as described in Usman et al., 1987,J. Am. Chem. Soc., 109, 7845; Scaringe et al., 1990, Nucleic Acids Res.,18, 5433; Wincott et al., 1995, Nucleic Acids Res. 23, 2677-2684; andWincott et al., 1997, Methods Mol. Bio., 74, 59, and may make use ofcommon nucleic acid protecting and coupling groups, such asdimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end. Themodified (e.g. 2′-O-methylated) nucleotides and unmodified nucleotidesare incorporated as desired.

Alternatively, the nucleic acid molecules of the present invention canbe synthesized separately and joined together post-synthetically, forexample, by ligation (Moore et al., 1992, Science 256, 9923; Draper etal., International PCT publication No. WO93/23569; Shabarova et al.,1991, Nucleic Acids Research 19, 4247; Bellon et al., 1997, Nucleosides& Nucleotides, 16, 951; Bellon et al., 1997, Bioconjugate Chem. 8, 204),or by hybridization following synthesis and/or deprotection.

The siRNA molecules of the invention can also be synthesized via atandem synthesis methodology, as described in US patent applicationpublication No. US2004/0019001 (McSwiggen) wherein both siRNA strandsare synthesized as a single contiguous oligonucleotide fragment orstrand separated by a cleavable linker which is subsequently cleaved toprovide separate siRNA fragments or strands that hybridize and permitpurification of the siRNA duplex. The linker can be a polynucleotidelinker or a non-nucleotide linker.

For further information, see PCT publication No. WO 2004/015107(ATUGEN).

As described above, the siRNAs of Table A (below) were constructed suchthat alternate sugars have 2′-O-methyl modification i.e. alternatenucleotides were thus modified. In these preferred embodiments, in onestrand of the siRNA the modified nucleotides were numbers 1, 3, 5, 7, 9,11, 13, 15, 17 and 19 and in the opposite strand the modifiednucleotides were numbers 2, 4, 6, 8, 10, 12, 14, 16 and 18. Thus thesesiRNAs are blunt-ended 19-mer RNA molecules with alternate 2-0′-methylmodifications as described above. The siRNAs of Tables 2 and 3 (below)are also constructed in this manner; the siRNAs of Table B areblunt-ended 19-mer RNA molecules with alternate 2-0′-methylmodifications; the siRNAs of Table C are blunt-ended 21-mer RNAmolecules with alternate 2-0′-methyl modifications.

Table A details various novel siRNA molecules which were generated andsubsequently synthesized for gene RTP801. The two final columns indicatethe results of two experiments performed to examine the activity of thenovel molecules. Briefly, HeLa or Hacat cells were transfected with aspecific novel siRNA to be tested. Expression of the RTP801 polypeptidewas then determined by western blotting using an antibody against theRTP801 polypeptide. Referring to the two right-hand columns of Table A,“−” signifies an inactive or low-activity molecule (which does notsubstantially inhibit the expression of the RTP801 gene); “+” signifiesan siRNA molecule with some inhibitory activity (of RTP801 geneexpression), “++” signifies a molecule with higher inhibitory activity,and so on. Any one of the the siRNA molecules disclosed herein, and inparticular the active molecules detailed in Table A are novel and alsoconsidered a part of the present invention. TABLE A No ID Name ORG POSAS (5′ −> 3′) SS (5′ −> 3′) HeLaB, 20 nM HaCat, 20 nM  1 REDD1 h 5′UTR128 UAGAAGCCGCAGCUAGCGC GCGCUAGCUGCGGCUUCUA + +  2 REDD2 hmr CDS 337UCCGAGCUCUCCAGGCUCG CGAGCCUGGAGAGCUCGGA − −  3 REDD3 hmr CDS 360UGCUGCUGUCCAGGGACUC GAGUCCCUGGACAGCAGCA − −  4 REDD4 hmr CDS 478AGCAGCUGCAUCAGGUUGG CCAACCUGAUGCAGCUGCU − −  5 REDD5 h CDS 728UGAGUCCAGGCGCAGCACG CGUGCUGCGCCUGGACUCA − −  6 Redd6 hmr 5′UTR 119CAGCUAGCGCGGUCAGCGA UCGCUGACCGCGCUAGCUG − −  7 Redd7 hmr 5′UTR 122CCGCAGCUAGCGCGGUCAG CUACCGCGCUAGCUGCGG − −  8 Redd8 hmr 5′UTR 125AAGCCGCAGCUAGCGCGGU ACCGCGCUAGCUGCGGCUU − −  9 Redd9 hmr CDS 339AGUCCGAGCUCUCCAGGCU AGCCUGGAGAGCUCGGACU − − 10 Redd10 hmr CDS 341GCAGUCCGAGCUCUCCAGG CCUGGAGAGCUCGGACUGC − − 11 Redd11 hmr CDS 363UGUUGCUGCUGUCCAGGGA UCCCUGGACAGCAGCAACA − − 12 Redd12 hmr CDS 369AGCCACUGUUGCUGCUGUC GACAGCAGCAACAGUGGCD − − 13 Redd13 hmr CDS 370AAGCCACUGUUGCUGCUGU ACAGCAGCAACAGUGGCUU − − 14 Redd14 hmr CDS 475AGCUGCAUCAGGUUGGCAC GUGCCAACCUGAUGCAGCU +++ +++ 15 Redd15 hmr CDS 481UGCAGCAGCUGCAUCAGGU ACCUGAUGCAGCUGCUGCA + + 16 Redd16 hmr CDS 486UCUCCUGCAGCAGCUGCAU AUGCAGCUGCUGCAGGAGA − − 17 Redd17 hmr CDS 610CCCCGCAGGCCGCACGGCU AGCCGUGCGGCCUGCGGGG − − 18 Redd18 hmr CDS 750CCUGGAUCUUGGGCCAGAG CUCUGGCCCAAGAUCCAGG − − 19 Redd19 hmr CDS 809CAGCGUCAGGGACUGGCUG CAGCCAGUCCCUGACGCUG − − 20 Redd20 hmr 3′UTR 1097 AUGCUACAGUACUGAGGGG CCCCUCAGUACUGUAGCAU + + 21 Redd21 hmr 3′UTR 1419 GUCUGUAAGAUAGCUGCCU AGGCAGCUAUCUUACAGAC + + 22 Redd22 hmr 3′UTR 1617 UUCUAGAUGGAAGACCCAG CUGGGUCUUCCAUCUAGAA ++ ++ 23 Redd23 hmr 3′UTR 1670 UUGAACAUCAAGUGUAUUC GAAUACACUUGAUGUUCAA ++ ++ 24 Redd24 hmr 3′UTR 1693 AAAUAUUGCAUAGGUUUA UAAGACCUAUGCAAUAUUU + + 25 Redd25 hmr 3′UTR 1695 AAAAAUAUUGCAUAGGUCU AGACCUAUGCAAUAUUUUU ++ ++ 26 Redd26 hmr CDS 349AGGGACUCGCAGUCCGAGC GCUCGGACUGCGAGUCCCU − − 27 Redd27 hmr 3′UTR 1673 UACUUGAACAUCAAGUGUA UACACUUGAUGUUCAAGUA ++ ++ 28 Redd28 hmr 3′UTR 1717 AAACAUGUUUAUUAGAAAA UUUUCUAAUAAACAUGUUU − − 29 Redd29 h 5′UTR  99AACUGCUAAGACAAGUGCG CGCACUUGUCUUAGCAGUU − − 30 Redd30 h CDS 213ACGACGACGAGAAGCGGUC GACCGCUUCUCGUCGUCGU − − 31 Redd31 h CDS 393AAGCCGUGUCUUCCUCCGG CCGGAGGAAGACACGGCUU − − 32 Redd32 h CDS 453AGUGUUCAUCCUCAGGGUC GACCCUGAGGAUGAACACU − − 33 Redd33 h CDS 521AGGGCGUCGAGAGCCCAGC GCUGGGCUCUCGACGCCCU − − 34 Redd34 hr CDS 535AUCAGCAGGCGCGCAGGGC GCCCUGCGCGCCUGCUGAU − − 35 Redd35 h CDS 571AGUUCUUUGCCCACCUGGC GCCAGGUGGGCAAAGAACU − − 36 Redd36 h CDS 597ACGGCUCGCUGUAGGCCAG CUGGCCUACAGCGAGCCGU − − 37 Redd37 h CDS 625ACGUCCAGCAGCGCCCCCC GGGGGGCGCUGCUGGACGU − − 38 Redd38 h CDS 829AUGACUCGGAAGCCAGUGC GCACUGGCUUCCGAGUCAU − − 39 Redd39 h 3′UTR 1046 AACUCAAUGAGCUUCCUGG CCAGGAAGCUCAUUGAGUU ++ ++ 40 REDD40 h 3′UTR 1539 CUCAACUCUGCAGUACACG CGUGUACUGCAGAGUUGAG + + 41 Redd41 h 3′UTR 1317 AGAUACACAAACCACCUCC GGAGGUGGUUUGUGUAUCU + + 42 Redd42 h 3′UTR 1350 ACAACAAACACACUUGGUC GACCAAGUGUGUUUGUUGU ++ ++ 43 Redd43 hmr CDS 473CUGCAUCAGGUUGGCACAC GUGUGCCAACCUGAUGCAG + + 44 REDD44 h 3′UTR 955UCCUGCCUCUAGUCUCCAC GUGGAGACUAGAGGCAGGA + + 45 Redd45 hmr CDS 476CAGCUGCAUCAGGUUGGCA UGCCAACCUGAUGCAGCUG − − 46 Redd46 hmr CDS 479CAGCAGCUGCAUCAGGUUG CAACCUGAUGCAGCUGCUG − − 47 Redd47 hmr CDS 483CCUGCAGCAGCUGCAUCAG CUGAUGCAGCUGCUGCAGG − − 48 Redd48 hmr CDS 485CUCCUGCAGCAGCUGCAUC GAUGCAGCUGCUGCAGGAG − − 49 REDD40.1 h 3′UTR 1536 AACUCUGCAGUACACGAUG CAUCGUGUACUGCAGAGUU ++ ++ 50 REDD44.1 h 3′UTR 954CCUGCCUCUAGUCUCCACC GGUGGAGACUAGAGGCAGG ++ ++

Note that in the above Table A, the sense strands of siRNAs 1-50 haveSEQ ID NOS: 3-52 respectively, and the antisense strands of siRNAs 1-50have SEQ ID NOS: 53-102 respectively. The molecule designated REDD 14has SEQ ID Nos 16 (Sense strand) and 66 (antisense strand). TABLE BOligo No Source Length Sense Sirna AntiSense Sirna 51 Human 19CTAGCCAGTTGGTAAGCCA TGGCTTACCAACTGGCTAG 52 Human 19 TGATTCCAGTGGTTGGAAATTTCCAACCACTGGAATCA 53 Human 19 CCAGTGGTTGGAAAACTGA TCAGTTTTCCAACCACTGG54 Human 19 GCTTCCGAGTCATCAAGAA TTCTTGATGACTCGGAAGC 55 Human 19GGAAGCTCATTGAGTTGTG CACAACTCAATGAGCTTCC 56 Human, cynomoglus 19CCATCTGGGTCTTCCATCT AGATGGAAGACCCAGATGG 57 Human, cynomoglus 19GGATGTGTGTGTAGCATGT ACATGCTACACACACATCC 58 Human, cynomoglus 19ACACATACCCCTCAGTACT AGTACTGAGGGGTATGTGT 59 Human, cynomoglus 19ACATACCCCTCAGTACTGT ACAGTACTGAGGGGTATGT 60 Human, cynomoglus 19CACTGTTCATGAATACACT AGTGTATTCATGAACAGTG 61 Human, cynomoglus 19CCAGCTGGATGTGTGTGTA TACACACACATCCAGCTGG 62 Human, cynomoglus 19CGGAACAGCTGCTCATTGA TCAATGAGCAGCTGTTCCG 63 Human, cynomoglus 19GAAGCTCATTGAGTTGTGT ACACAACTCAATGAGCTTC 64 Human, cynomoglus 19GGACACATACCCCTCAGTA TACTGAGGGGTATGTGTCC 65 Human, cynomoglus 19GGATCTTTGACACTTGAAA TTTCAAGTGTCAAAGATCC 66 Human, cynomoglus 19GTAGCATGTACCTTATTAT ATAATAAGGTACATGCTAC 67 Human, cynomoglus 19TCAGTACTGTAGCATGGAA TTCCATGCTACAGTACTGA 68 Human, cynomoglus 19TGTGTAGCATGTACCTTAT ATAAGGTACATGCTACACA 69 Human, cynomoglus 19CTGGATGTGTGTGTAGCAT ATGCTACACACACATCCAG 70 Human, cynomoglus, mouse 19ACACTTGATGTTCAAGTAT ATACTTGAACATCAAGTGT 71 Human, cynomoglus, mouse 19GCATGAATGTAAGAGTAGG CCTACTCTTACATTCATGC 72 Human, cynomoglus, mouse 19AGCAGCAACAGTGGCTTCG CGAAGCCACTGTTGCTGCT 73 Human, cynomoglus, mouse 19ATGAATGTAAGAGTAGGAA TTCCTACTCTTACATTCAT 74 Human, cynomoglus, mouse 19CAGCAGCAACAGTGGCTTC GAAGCCACTGTTGCTGCTG 75 Human, cynomoglus, mouse 19CATGAATGTAAGAGTAGGA TCCTACTCTTACATTCATG 76 Human, cynomoglus, mouse 19GATGTTCAAGTATTAAGAC GTCTTAATACTTGAACATC 77 Human, cynomoglus, mouse, rat19 TGATGCAGCTGCTGCAGGA TCCTGCAGCAGCTGCATCA 78 Human, cynomoglus, mouse,rat 19 GAATACACTTGATGTTCAA TTGAACATCAAGTGTATTC 79 Human, cynomoglus,mouse, rat 19 TGAATACACTTGATGTTCA TGAACATCAAGTGTATTCA 80 Human,cynomoglus, mouse, rat 19 ATACACTTGATGTTCAAGT ACTTGAACATCAAGTGTAT 81Human, cynomoglus, mouse, rat 19 CATGAATACACTTGATGTT AACATCAAGTGTATTCATG82 Human, cynomoglus, mouse, rat 19 CTGGACAGCAGCAACAGTGCACTGTTGCTGCTGTCCAG 83 Human, cynomoglus, mouse, rat 19GTTCATGAATACACTTGAT ATCAAGTGTATTCATGAAC 84 Human, cynomoglus, mouse, rat19 TCATGAATACACTTGATGT ACATCAAGTGTATTCATGA 85 Human, cynomoglus, mouse,rat 19 TGGACAGCAGCAACAGTGG CCACTGTTGCTGCTGTCCA 86 Human, cynomoglus,mouse, rat 19 TGTGTGCCAACCTGATGCA TGCATCAGGTTGGCACACA 87 Human,cynomoglus, mouse, rat 19 TTCATGAATACACTTGATG CATCAAGTGTATTCATGAA 88Human, cynomoglus, mouse, rat 19 AACCTGATGCAGCTGCTGC GCAGCAGCTGCATCAGGTT89 Human, cynomoglus, mouse, rat 19 AGTCCCTGGACAGCAGCAATTGCTGCTGTCCAGGGACT 90 Human, cynomoglus, mouse, rat 19CCCTCAGTACTGTAGCATG CATGCTACAGTACTGAGGG 91 Human, cynomoglus, mouse, rat19 CCTGGACAGCAGCAACAGT ACTGTTGCTGCTGTCCAGG 92 Human, cynomoglus, mouse,rat 19 TGTGCCAACCTGATGCAGC GCTGCATCAGGTTGGCACA 93 Human, cynomoglus,mouse, rat 19 AATACACTTGATGTTCAAG CTTGAACATCAAGTGTATT 94 Human,cynomoglus, mouse, rat 19 ATGAATACACTTGATGTTC GAACATCAAGTGTATTCAT 95Human, cynomoglus, rat 19 TGATGCAGCTGCTGCAGGA TCCTGCAGCAGCTGCATCA 96Human, cynomoglus, rat 19 AGAACTGTTTACATGAAGA TCTTCATGTAAACAGTTCT 97Human, cynomoglus, rat 19 ATCTAGAACTGTTTACATG CATGTAAACAGTTCTAGAT 98Human, cynomoglus, rat 19 CCATGCCTAGCCTTTGGGA TCCCAAAGGCTAGGCATGG 99Human, cynomoglus, rat 19 CTAGAACTGTTTACATGAA TTCATGTAAACAGTTCTAG 100 Human, cynomoglus, rat 19 GAACTGTTTACATGAAGAT ATCTTCATGTAAACAGTTC 101 Human, cynomoglus, rat 19 GGTCTTCCATCTAGAACTG CAGTTCTAGATGGAAGACC 102 Human, cynomoglus, rat 19 CCATCTAGAACTGTTTACA TGTAAACAGTTCTAGATGG 103 Human, cynomoglus, rat 19 CTTCCATCTAGAACTGTTT AAACAGTTCTAGATGGAAG 104 Human, cynomoglus, rat 19 TAGAACTGTTTACATGAAG CTTCATGTAAACAGTTCTA 105 Human, cynomoglus, rat 19 TCTTCCATCTAGAACTGTT AACAGTTCTAGATGGAAGA 106 Human, cynomoglus, rat 19 CATCTAGAACTGTTTACAT ATGTAAACAGTTCTAGATG 107 Human, cynomoglus, rat 19 GGGTCTTCCATCTAGAACT AGTTCTAGATGGAAGACCC 108 Human, cynomoglus, rat 19 TCCATCTAGAACTGTTTAC GTAAACAGTTCTAGATGGA 109 Human, cynomoglus, rat 19 TCTAGAACTGTTTACATGA TCATGTAAACAGTTCTAGA 110 Human, cynomoglus, rat 19 TTCCATCTAGAACTGTTTA TAAACAGTTCTAGATGGAA 111 Human, cynomoglus, rat 19 GTCTTCCATCTAGAACTGT ACAGTTCTAGATGGAAGAC 112 Human, mouse 19 CAAGTATTAAGACCTATGC GCATAGGTCTTAATACTTG 113  Human,mouse 19 GTATTAAGACCTATGCAAT ATTGCATAGGTCTTAATAC 114  Human, mouse 19AGTATTAAGACCTATGCAA TTGCATAGGTCTTAATACT 115  Human, mouse 19ATGTTCAAGTATTAAGACC GGTCTTAATACTTGAACAT 116  Human, mouse 19CACTTGATGTTCAAGTATT AATACTTGAACATCAAGTG 117  Human, mouse 19CCAAGATCCAGGGGCTGTT AACAGCCCCTGGATCTTGG 118  Human, mouse 19GTTCAAGTATTAAGACCTA TAGGTCTTAATACTTGAAC 119  Human, mouse 19TCAAGTATTAAGACCTATG CATAGGTCTTAATACTTGA 120  Human, mouse 19AAGTATTAAGACCTATGCA TGCATAGGTCTTAATACTT 121  Human, mouse 19TGTTCAAGTATTAAGACCT AGGTCTTAATACTTGAACA 122  Human, mouse, rat 19TGGGTCTTCCATCTAGAAC GTTCTAGATGCAAGACCCA gi9506686ref gi21312867refgi18376838ref Overlap NM_019058.1 NM_029083.1 NM_080906.1 with pet-1 No(Homo sapiens) (Mouse) (Rat) (antisense)* 51 [556-574] − − − 52 [984-1002] − − − 53  [989-1007] − − − 54 [835-853] [763-781] − − 55[1049-1067] − − 56 [1613-1631] [1569-1583] [1610-1624] + 57 [1152-1170]− − − 58 [1090-1108] [1081-1098] − 59 [1092-1110] [1082-1100] − 60[1660-1678] [1612-1626] [1652-1666] + 61 [1146-1164] [1099-1114][1139-1154] − 62 [868-886] [801-814] [854-867] − 63 [1050-1068] − − − 64[1088-1106] − − − 65 [1483-1501] [1424-1442] − − 66 [1162-1180][1112-1118] − − 67 [1101-1119] [1091-1106] − 68 [1159-1177] [1111-1127][1151-1167] − 69 [1150-1168] − − − 70 [1674-1692] [1622-1640] + 71[1438-1456] [1379-1397] − 72 [372-390] [300-318] − 73 [1440-1458][1381-1399] − 74 [371-389] [299-317] − 75 [1439-1457] [1380-1398] − 76[1680-1698] [1628-1646] + 77 [484-502] [412-430] [465-483] − 78[1670-1688] [1618-1636] + 79 [1669-1687] [1617-1635] [1657-1675] + 80[1672-1690] [1620-1638] [1660-1678] + 81 [1667-1685] [1615-1633][1655-1673] + 82 [366-384] [294-312] [347-365] − 83 [1664-1682][1612-1630] [1652-1670] + 84 [1666-1684] [1614-1632] [1654-1672] + 85[367-385] [295-313] [348-366] − 86 [472-490] [400-418] [453-471] − 87[1665-1683] [1613-1631] [1653-1671] + 88 [480-498] [408-426] [461-479] −89 [361-379] [289-307] [342-360] − 90 [1098-1116] [1048-1066][1088-1106] − 91 [365-383] [293-311] [346-364] − 92 [474-492] [402-420][455-473] − 93 [1671-1689] [1619-1637] [1659-1677] + 94 [1668-1686][1616-1634] [1656-1674] − 95 [484-502] [465-483] + 96 [1632-1650][1625-1643] + 97 [1628-1646] [1621-1639] + 98 [196-214] [186-204] − 99[1630-1648] [1623-1641] + 100  [1633-1651] [1626-1644] + 101 [1620-1638] [1613-1631] + 102  [1626-1644] [1619-1637] + 103 [1623-1641] [1616-1634] + 104  [1631-1649] [1624-1642] + 105 [1622-1640] [1615-1633] + 106  [1627-1645] [1620-1638] + 107 [1619-1637] [1612-1630] + 108  [1625-1643] [1618-1636] + 109 [1629-1647] [1622-1640] + 110  [1624-1642] [1617-1635] + 111 [1621-1639] [1614-1632] + 112  [1686-1702] [1634-1652] + 113 [1689-1707] [1637-1655] + 114  [1688-1706] [1636-1654] + 115 [1681-1699] [1629-1647] + 116  [1675-1693] [1623-1641] + 117  [757-775][685-703] − 118  [1683-1701] [1631-1649] + 119  [1685-1703][1633-1651] + 120  [1687-1705] [1635-1653] + 121  [1682-1700][1630-1648] + 122  [1618-1636] [1570-1588] [1611-1629] +

Note that in the above Table B, the sense strands of siRNAs 51-122 haveSEQ ID NOS: 103-174 respectively, and the antisense strands of siRNAs51-122 have SEQ ID NOS: 175-246 respectively. TABLE C Oligo No SourceLength Sense Sirna AntiSense Sirna 123 Human 21 CCAGGAAGCTCATTGAGTTGTACAACTCAATGAGCTTCCTGG 124 Human 21 CCATCTGGGTCTTCCATCTAGCTAGATGGAAGACCCAGATGG 125 Human 21 GGATGTGTGTGTAGCATGTACGTACATGCTACACACACATCC 126 Human 21 CAAGTGTGTTTGTTGTTTGTTAACAAACAACAAACACACTTG 127 Human 21 CCTCAGTACTGTAGCATGGAATTCCATGCTACAGTACTGAGG 128 Human 21 GACCAAGTGTGTTTGTTGTTTAAACAACAAACACACTTGGTC 129 Human 21 GCTTCCGAGTCATCAAGAAGATCTTCTTGATGACTCGGAAGC 130 Human 21 GGAGGTGGGGGAATAGTGTTTAAACACTATTCCCCCACCTCC 131 Human 21 CAGTACTGTAGCATGGAACAATTGTTCCATGCTACAGTACTG 132 Human, cynomoglus 21 GAATACACTTGATGTTCAAGTACTTGAACATCAAGTGTATTC 133 Human, cynomoglus 21 CAAGTATTAAGACCTATGCAATTGCATAGGTCTTAATACTTG 134 Human, cynomoglus 21 GAACTTTTGGGGTGGAGACTATAGTCTCCACCCCAAAAGTTC 135 Human, cynomoglus 21 GGACACATACCCCTCAGTACTAGTACTGAGGGGTATGTGTCC 136 Human, cynomoglus 21 GGAGGTGGTTTGTGTATCTTATAAGATACACAAACCACCTCC 137 Human, cynomoglus 21 GGATCTTTGACACTTGAAAAATTTTTCAAGTGTCAAAGATCC 138 Human, cynomoglus 21 GGTCTTCCATCTAGAACTGTTAACAGTTCTAGATGGAAGACC 139 Human, cynomoglus 21 TGTGTAGCATGTACCTTATTATAATAAGGTACATGCTACACA 140 Human, cynomoglus 21 CAACAAGGCTTCCAGCTGGATATCCAGCTGGAAGCCTTGTTG 141 Human, cynomoglus 21 CACTTGGGATCTTTGACACTTAAGTGTCAAAGATCCCAAGTG 142 Human, cynomoglus 21 CATCACTACTGACCTGTTGTATACAACAGGTCAGTAGTGATG 143 Human, cynomoglus 21 GTGTGTGTAGCATGTACCTTATAAGGTACATGCTACACACAC 144 Human, cynomoglus, mouse 21GCATGAATGTAAGAGTAGGAA TTCCTACTCTTACATTCATGC 145 Human, cynomoglus, mouse21 GACAGCAGCAACAGTGGCTTC GAAGCCACTGTTGCTGCTGTC 146 Human, cynomoglus,mouse, rat 21 TGATGCAGCTGCTGCAGGAGA TCTCCTGCAGCAGCTGCATCA 147 Human,cynomoglus, mouse, rat 21 TGAATACACTTGATGTTCAAG CTTGAACATCAAGTGTATTCA148 Human, cynomoglus, mouse, rat 21 CATGAATACACTTGATGTTCATGAACATCAAGTGTATTCATG 149 Human, cynomoglus, mouse, rat 21GGACAGCAGCAACAGTGGCTT AAGCCACTGTTGCTGCTGTCC 150 Human, cynomoglus,mouse, rat 21 GTTCATGAATACACTTGATGT ACATCAAGTGTATTCATGAAC 151 Human,cynomoglus, mouse, rat 21 TCATGAATACACTTGATGTTC GAACATCAAGTGTATTCATGA152 Human, cynomoglus, mouse, rat 21 TCCCTGGACAGCAGCAACAGTACTGTTGCTGCTGTCCAGGGA 153 Human, cynomoglus, mouse, rat 21AGTCCCTGGACAGCAGCAACA TGTTGCTGCTGTCCAGGGACT 154 Human, cynomoglus, rat21 GAATACACTTGATGTTCAAGT ACTTGAACATCAAGTGTATTC 155 Human, cynomoglus,rat 21 CTAGAACTGTTTACATGAAGA TCTTCATGTAAACAGTTCTAG 156 Human,cynomogius, rat 21 CCATCTAGAACTGTTTACATG CATGTAAACAGTTCTAGATGG 157Human, cynomoglus, rat 21 CTTCCATCTAGAACTGTTTAC GTAAACAGTTCTAGATGGAAG158 Human, cynomoglus, rat 21 TCTTCCATCTAGAACTGTTTATAAACAGTTCTAGATGGAAGA 159 Human, cynomoglus, rat 21CATCTAGAACTGTTTACATGA TCATGTAAACAGTTCTAGATG 160 Human, cynomoglus, rat21 GGGTCTTCCATCTAGAACTGT ACAGTTCTAGATGGAAGACCC 161 Human, cynomoglus,rat 21 TCCATCTAGAACTGTTTACAT ATGTAAACAGTTCTAGATGGA 162 Human,cynomoglus, rat 21 TCTAGAACTGTTTACATGAAG CTTCATGTAAACAGTTCTAGA 163Human, cynomoglus, rat 21 TTCCATCTAGAACTGTTTACA TGTAAACAGTTCTAGATGGAA164 Human, cynomoglus, rat 21 GTCTTCCATCTAGAACTGTTTAAACAGTTCTAGATGGAAGAC 165 Human, mouse 21 TGATGTTCAAGTATTAAGACCGGTCTTAATACTTGAACATCA 166 Human, mouse 21 GTTCAAGTATTAAGACCTATGCATAGGTCTTAATACTTGAAC 167 Human, mouse 21 TCAAGTATTAAGACCTATGCATGCATAGGTCTTAATACTTGA 168 Human, mouse 21 GATGTTCAAGTATTAAGACCTAGGTCTTAATACTTGAACATC 169 Human, mouse 21 TTCAAGTATTAAGACCTATGCGCATAGGTCTTAATACTTGAA 170 Human, rat 21 CTGGGTCTTCCATCTAGAACTAGTTCTAGATGGAAGACCCAG 171 Human, rat 21 TGGGTCTTCCATCTAGAACTGCAGTTCTAGATGGAAGACCCA gi9506686ref gi21312867ref gi18376838ref OverlapNM_019058.1 fNM_029083.1 fNM_080906.1 with pet-1 No (Homo sapiens) (Musmusculus) (Rattus norvegicus) (antisense) 123 [1046-1066] − − − 124[1613-1633] [1569-1585] [1610-1626] + 125 [1152-1172] [1102-1122][1142-1161] − 126 [1353-1373] − − − 127 [1099-1119] [1049-1066][1089-1106] − 128 [1350-1370] − − − 129 [835-855] [763-783] − − 130[1024-1044] [976-986] − − 131 [1102-1122] [1052-1072] − 132 [1670-1690][1618-1638] [1658-1678] + 133 [1686-1706] [1634-1654] [1674-1694] + 134[944-964] − − − 135 [1088-1108] [1047-1058] [1081-1098] − 136[1317-1337] [1256-1268] − − 137 [1483-1503] [1424-1442] − − 138[1620-1640] [1572-1588] [1613-1633] + 139 [1159-1179] [1111-1128][1151-1169] − 140 [1135-1155] − − 141 [1477-1497] − − − 142 [1399-1419][1341-1356] [1383-1398] − 143 [1156-1176] [1106-1126] [1146-1166] − 144[1438-1458] [1379-1399] − 145 [369-389] [297-317] − 146 [484-504][412-432] [465-485] − 147 [1669-1689] [1617-1637] [1657-1677] + 148[1667-1687] [1615-1635] [1655-1675] + 149 [368-388] [296-316] [349-369]− 150 [1664-1684] [1612-1632] [1652-1672] + 151 [166-1686] [1614-1634][1654-1674] + 152 [363-383] [291-311] [344-364] − 153 [361-381][289-309] [342-362] − 154 [1670-1690] [1658-1679] + 155 [1630-1650][1623-1643] + 156 [1626-1646] [1619-1639] + 157 [1623-1643][1616-1636] + 158 [1622-1642] [1615-1635] + 159 [1627-1647][1620-1640] + 160 [1619-1639] [1612-1632] + 161 [1625-1645][1618-1638] + 162 [1629-1649] [1622-1642] + 163 [1624-1644][1617-1637] + 164 [1621-1641] [1614-1634] + 165 [1679-1699][1627-1647] + 166 [1683-1703] [1631-1651] + 167 [1685-1705][1633-1653] + 168 [1680-1700] [1628-1648] + 169 [1684-1704][1632-1652] + 170 [1617-1637] [1610-1630] + 171 [1618-1638] [1611-1631]+

Note that in the above Table C, the sense strands of siRNAs 123-171 haveSEQ ID NOS: 247-295 respectively, and the antisense strands of siRNAs123-171 have SEQ ID NOS: 296-344 respectively.

Example 10

Pharmacology and Drug Delivery

The nucleotide sequences of the present invention can be deliveredeither directly or with viral or non-viral vectors. When delivereddirectly the sequences are generally rendered nuclease resistant.Alternatively the sequences can be incorporated into expressioncassettes or constructs such that the sequence is expressed in the cellas discussed herein below. Generally the construct contains the properregulatory sequence or promoter to allow the sequence to be expressed inthe targeted cell.

The compounds or pharmaceutical compositions of the present inventionare administered and dosed in accordance with good medical practice,taking into account the clinical condition of the individual patient,the disease to be treated, the site and method of administration,scheduling of administration, patient age, sex, body weight and otherfactors known to medical practitioners.

The pharmaceutically “effective amount” for purposes herein is thusdetermined by such considerations as are known in the art. The amountmust be effective to achieve improvement including but not limited toimproved survival rate or more rapid recovery, or improvement orelimination of symptoms and other indicators as are selected asappropriate measures by those skilled in the art.

The treatment generally has a length proportional to the length of thedisease process and drug effectiveness and the patient species beingtreated. It is noted that humans are treated generally longer than themice or other experimental animals exemplified herein.

The compounds of the present invention can be administered by any of theconventional routes of administration. It should be noted that thecompound can be administered as the compound or as pharmaceuticallyacceptable salt and can be administered alone or as an active ingredientin combination with pharmaceutically acceptable carriers, solvents,diluents, excipients, adjuvants and vehicles. The compounds can beadministered orally, subcutaneously or parenterally includingintravenous, intraarterial, intramuscular, intraperitoneally, andintranasal administration as well as intrathecal and infusiontechniques. Implants of the compounds are also useful. Liquid forms maybe prepared for injection, the term including subcutaneous, transdermal,intravenous, intramuscular, intrathecal, and other parental routes ofadministration. The liquid compositions include aqueous solutions, withand without organic cosolvents, aqueous or oil suspensions, emulsionswith edible oils, as well as similar pharmaceutical vehicles. Inaddition, under certain circumstances the compositions for use in thenovel treatments of the present invention may be formed as aerosols, forintranasal and like administration. The patient being treated is awarm-blooded animal and, in particular, mammals including man. Thepharmaceutically acceptable carriers, solvents, diluents, excipients,adjuvants and vehicles as well as implant carriers generally refer toinert, non-toxic solid or liquid fillers, diluents or encapsulatingmaterial not reacting with the active ingredients of the invention.

When administering the compound of the present invention parenterally,it is generally formulated in a unit dosage injectable form (solution,suspension, emulsion). The pharmaceutical formulations suitable forinjection include sterile aqueous solutions or dispersions and sterilepowders for reconstitution into sterile injectable solutions ordispersions. The carrier can be a solvent or dispersing mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, liquid polyethylene glycol, and the like), suitablemixtures thereof, and vegetable oils.

Proper fluidity can be maintained, for example, by the use of a coatingsuch as lecithin, by the maintenance of the required particle size inthe case of dispersion and by the use of surfactants. Nonaqueousvehicles such a cottonseed oil, sesame oil, olive oil, soybean oil, cornoil, sunflower oil, or peanut oil and esters, such as isopropylmyristate, can also be used as solvent systems for compoundcompositions. Additionally, various additives which enhance thestability, sterility, and isotonicity of the compositions, includingantimicrobial preservatives, antioxidants, chelating agents, andbuffers, can be added. Prevention of the action of microorganisms can beensured by various antibacterial and antifungal agents, for example,parabens, chlorobutanol, phenol, sorbic acid, and the like. In manycases, it is desirable to include isotonic agents, for example, sugars,sodium chloride, and the like. Prolonged absorption of the injectablepharmaceutical form can be brought about by the use of agents delayingabsorption, for example, aluminum monostearate and gelatin. According tothe present invention, however, any vehicle, diluent, or additive usedhave to be compatible with the compounds.

Sterile injectable solutions can be prepared by incorporating thecompounds utilized in practicing the present invention in the requiredamount of the appropriate solvent with various of the other ingredients,as desired.

A pharmacological formulation of the present invention can beadministered to the patient in an injectable formulation containing anycompatible carrier, such as various vehicle, adjuvants, additives, anddiluents; or the compounds utilized in the present invention can beadministered parenterally to the patient in the form of slow-releasesubcutaneous implants or targeted delivery systems such as monoclonalantibodies, vectored delivery, iontophoretic, polymer matrices,liposomes, and microspheres. Examples of delivery systems useful in thepresent invention include U.S. Pat. Nos. 5,225,182; 5,169,383;5,167,616; 4,959,217; 4,925,678; 4,487,603; 4,486,194; 4,447,233;4,447,224; 4,439,196; and 4,475,196. Many other such implants, deliverysystems, and modules are well known to those skilled in the art.

A pharmacological formulation of the compound utilized in the presentinvention can be administered orally to the patient. Conventionalmethods such as administering the compound in tablets, suspensions,solutions, emulsions, capsules, powders, syrups and the like are usable.Known techniques which deliver it orally or intravenously and retain thebiological activity are preferred. In one embodiment, the compound ofthe present invention can be administered initially by intravenousinjection to bring blood levels to a suitable level. The patient'slevels are then maintained by an oral dosage form, although other formsof administration, dependent upon the patient's condition and asindicated above, can be used.

In general, the active dose of compound for humans is in the range offrom 1 ng/kg to about 20-100 mg/kg body weight per day, preferably about0.01 mg to about 2-10 mg/kg body weight per day, in a regimen of onedose per day or twice or three or more times per day for a period of 1-2weeks or longer, preferably for 24- to 48 hrs or by continuous infusionduring a period of 1-2 weeks or longer.

Administration of Compounds of the Present Invention to the Eye

The compounds of the present invention can be administered to the eyetopically or in the form of an injection, such as an intravitrealinjection, a sub-retinal injection or a bilateral injection. Furtherinformation on administration of the compounds of the present inventioncan be found in Tolentino et al., Retina 24 (2004) 132-138; Reich etal., Molecular vision 9 (2003) 210-216.

Pulmonary Administration of Compounds of the Present Invention

The therapeutic compositions of the present invention are preferablyadministered into the lung by inhalation of an aerosol containing thesecompositions/compounds, or by intranasal or intratracheal instillationof said compositions. Formulating the compositions in liposomes maybenefit absorption. Additionally, the compositions may include a PFCliquid such as perflubron, and the compositions may be formulated as acomplex of the compounds of the invention with polyethylemeimine (PEI).

For further information on pulmonary delivery of pharmaceuticalcompositions see Weiss et al., Human gene therapy 10:2287-2293 (1999);Densmore et al., Molecular therapy 1:180-188 (1999); Gautam et al.,Molecular therapy 3:551-556 (2001); and Shahiwala & Misra, AAPSPharmSciTech 5 (2004). Additionally, respiratory formulations for siRNAare described in U.S. patent application No. 2004/0063654 of Davis etel.

Additional formulations for improved delivery of the compounds of thepresent invention can include non-formulated compounds, compoundscovalently bound to cholesterol, and compounds bound to targetingantibodies (Song et al., Antibody mediated in vivo delivery of smallinterfering RNAs via cell-surface receptors, Nat Biotechnol. 2005 June;23(6):709-17).

1. A method of treating a patient suffering from a respiratory disorder,an eye disease, a microvascular disorder, or a spinal cord injury ordisease which comprises administering to the patient a pharmaceuticalcomposition comprising an RTP801 inhibitor in an amount effective totreat the patient.
 2. A method according to claim 1, wherein the eyedisease is macular degeneration.
 3. A method according to claim 1,wherein the respiratory disorder is COPD.
 4. A method according to claim1, wherein the respiratory disorder is asthma.
 5. A method according toclaim 1, wherein the respiratory disorder is chronic bronchitis.
 6. Amethod according to claim 1, wherein the respiratory disorder isemphysema.
 7. A method according to claim 1, wherein the microvasculardisorder is diabetic retinopathy.
 8. A method according to claim 1,wherein the microvascular disorder is acute renal failure.
 9. The methodaccording to claim 1, wherein the inhibitor comprises a polynucleotidewhich comprises consecutive nucleotides having a sequence of sufficientlength and homology to a sequence present within the sequence of theRTP801 gene set forth in SEQ ID NO:1 to permit hybridization of theinhibitor to the gene.
 10. The method according to claim 1, wherein theinhibitor comprises an antibody which specifically binds to an epitopepresent within a polypeptide which comprises consecutive amino acids,the sequence of which is set forth in FIG. 2 (SEQ ID No:2).
 11. A methodfor treating a patient suffering from a respiratory disorder, an eyedisease, a microvascular disorder or a spinal cord injury or diseasewhich comprises administering to the patient a pharmaceuticalcomposition comprising a therapeutically effective amount of apolynucleotide RTP801 inhibitor so as to thereby treat the patient. 12.A method according to claim 11, wherein the polynucleotide is siRNA. 13.The method according to claim 12, wherein the siRNA comprisesconsecutive nucleotides having a sequence identical to any one of thesequences set forth in any one of Tables A-C (SEQ ID NOs:3-344).
 14. Themethod according to claim 11, wherein the inhibitor is selected from thegroup consisting of an siRNA molecule, a vector comprising an siRNAmolecule, a vector which can express an siRNA molecule and a moleculewhich is endogenously processed into an siRNA molecule.
 15. Use of atherapeutically effective amount of an RTP801 inhibitor for thepreparation of a medicament for promoting recovery in a patientsuffering from a respiratory disorder, an eye disease, a microvasculardisorder or spinal cord injury or disease.
 16. The use according toclaim 15, wherein the eye disease is macular degeneration.
 17. The useaccording to claim 15, wherein the respiratory disorder is COPD.
 18. Theuse according to claim 15, wherein the respiratory disorder is asthma.19. The use according to claim 15, wherein the respiratory disorder ischronic bronchitis.
 20. The use according to claim 15, wherein therespiratory disorder is emphysema.
 21. The use according to claim 15,wherein the microvascular disorder is diabetic retinopathy.
 22. The useaccording to claim 15, wherein the microvascular disorder is acute renalfailure.
 23. The use according to claim 15, wherein the inhibitorcomprises a polynucleotide which comprises consecutive nucleotideshaving a sequence of sufficient length and homology to a sequencepresent within the sequence of the RTP801 gene set forth in SEQ ID NO:1to permit hybridization of the inhibitor to the gene.
 24. The useaccording to claim 15, wherein the RTP801 inhibitor is an antibody whichspecifically binds to an epitope present within a polypeptide whichcomprises consecutive amino acids, the sequence of which is set forth inFIG. 2 (SEQ ID No:2).
 25. The use according to claim 15, wherein thepolynucleotide down regulates the expression of gene RTP801.
 26. The useaccording to claim 15, wherein the polynucleotide is an siRNA.
 27. Theuse according to claim 26, wherein the siRNA comprises consecutivenucleotides having a sequence identical to any one of the sequences setforth in any one of Tables A-C (SEQ ID NOs:3-344).
 28. The use accordingto claim 25, wherein the inhibitor is selected from the group consistingof an siRNA molecule, a vector comprising an siRNA molecule, a vectorwhich can express an siRNA molecule and a molecule which is endogenouslyprocessed into an siRNA molecule.